Methods for modulating angiogenesis

ABSTRACT

Recombinant plasminogen activator inhibitor-1 (PAI-1) isoforms which lack the reactive center loop and contain the complete heparin-binding domain or lack at least a portion of the heparin-binding domain are described. The rPAI-1 isoforms disclosed herein may be used to modulate angiogenesis through blocking release of VEGF from a VEGF-heparin complex. Furthermore, the rPAI-1 proteins may be used to inhibit cell proliferation and migration, induce apoptosis, and produce proteolytic fragments corresponding to angiostatin kringles 1-3 and kringles 1-4. A truncated proteolytic plasmin protein of 34 kDa is also provided.

INTRODUCTION

[0001] This application is a continuation-in-part of PCT/US03/09981,filed Apr. 1, 2003, and claims the benefit of priority from U.S.provisional application Serial No. 60/448,301, filed Feb. 14, 2003, andU.S. provisional application Serial No. 60/369,392, filed Apr. 1, 2002,whose contents are incorporated herein by reference in their entireties.This invention was supported in part by funds from the U.S. government(NIH NHLB1 Grant No. Rβ1-HL59590) and the U.S. government may thereforehave certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] Angiogenesis is the formation of new capillary blood vessels asoutgrowths of pre-existing vessels. The tightly regulated process playsa vital role in many physiological processes, such as embryogenesis,wound healing and menstruation. Angiogenesis is also important incertain pathological events. In addition to a role in solid tumor growthand metastasis, other notable conditions with an angiogenic componentare arthritis, psoriasis and diabetic retinopathy (Hanahan and Folkman(1996) Cell 86:353-364; Fidler and Ellis (1994) Cell 79(2):185-188).

[0003] At the onset of angiogenesis, the quiescent endothelium isdestabilized into migratory, proliferative endothelial cells. Theangiogenic (activated) endothelium is maintained primarily by positiveregulatory molecules. In the absence of such molecules, the endotheliumremains in a differentiated, quiescent state that is maintained bynegative regulatory molecules, angiogenesis inhibitors (Bouck (1990)Cancer Cells 2:179-185; Hanahan and Folkman (1996) supra). Normally, thenegative and positive activities are balanced to maintain the vascularendothelium in quiescence (Hanahan and Folkman (1996) supra; Folkman andKlagsbrum (1987) Science 235:442-447). A shift in the balance of thepositive and negative regulatory molecules can alter the differentiatedstate of the endothelium from the non-angiogenic, quiescent to theangiogenic state (Hanahan and Folkman (1996) supra). In the switch topro-angiogenesis, the quiescent endothelial cells are stimulated tomigrate toward a chemotactic stimulus, lining up in a tube (sprout)formation (Folkman and Klagsbrum (1987) supra). These cells also secreteproteolytic enzymes that degrade the endothelial basement membrane, thusallowing the migrating endothelial cells to extend into the perivascularstroma to begin a new capillary sprout. The angiogenic process ischaracterized by increased proliferation of endothelial cells to formthe extending capillary (Folkman and Klagsbrum (1987) supra; Moses, etal. (1995) Int. Rev. Cytol. 161:1-48; Martiny-Baron and Marme (1995)Curr. Opin. Biotechnol. 6:675-680; Liotta, et al. (1991) Cell64:327-336).

[0004] Vascular endothelial growth factor (VEGF) is a mitogenic factorthat stimulates pro-angiogenic properties, including endothelial cellmigration and proliferation. VEGF induces the expression of plasminogenactivator proteolytic pathway proteins that participate in cellularinvasive and remodeling processes (Pepper, et al. (1991) Biochem.Biophys. Res. Commun. 181:902-906; Mandriota, et al. (1995) J. Biol.Chem. 270:9709-9716; Mignatti, et al. (1989) J. Cell. Biol.108:671-682). VEGF-A RNA can undergo alternative splicing to producefour isoforms (Leung, et al. (1989) Science 246:1306-1309; Houck, et al.(1991) Mol. Endocriniology 5:1806-1814; Tischer, et al. (1991) J. Biol.Chem. 26611947-22954). Three of those isoforms, VEGF-A_(165,189,206),bind to heparin. Pro-VEGF affinity for heparin appears to be importantin the regulation of the availability of VEGF at the cell surface(Houck, et al. (1992) J. Biol. Chem. 267:26031-26037), where it caninteract with its tyrosine kinase receptors to exert its activity(Gitay-Goren, et al. (1992) J. Biol. Chem. 267:6093-6098). VEGF-A can bereleased from heparin in an inactive or active form (Ortega, et al.(1998) Biol. Cell 90:381-390). Plasmin and urokinase plasminogenactivator (uPA) cleaves pro-VEGF into an active form of varied sizesdepending upon the isoform and the activator molecule (Plouet, et al.(1997) J. Biol. Chem. 272:13390-13396).

[0005] There are naturally occurring molecules that serve as negativeregulators of angiogenesis. Angiostatin, one such negative regulator, isa 38-45 kDa cleavage product of plasminogen, containing kringle domains1-4 (K1-4) (O'Reilly, et al. (1994) Cell 79:315-328; O'Reilly, et al.(1996) Nat. Med. 2:689-692). Plasminogen, the precursor of plasmin, isactivated when it is cleaved at the carboxy-terminus by plasminogenactivators. The amino terminus contains five consecutive kringledomains, each approximately 9 kDa. The greatest inhibitory activity ofangiostatin is contained within kringles 1-3 (Cao, et al. (1996) J.Biol. Chem. 271:29461-29467) and kringles 1-5 (Cao (1999) Proc. Natl.Acad. Sci. USA 6:5728-5733). The mechanism for angiostatin inhibition ofendothelial cell growth in vitro and angiogenesis in vivo is unclear.

[0006] Plasminogen activator inhibitor-1 (PAI-1), a serpin family,serine protease inhibitor, is a multifunctional regulatory protein inthe plasminogen activator proteolytic (Chapman, et al. (1982) Cell28:653-662; Chapman (1997) Curr. Opin. Cell Biol. 9:714-724) andfibrinolytic pathways (Loskutoff and Curriden (1990) Ann. NY Acad. Sci.598:238-247; Collen (1999) Thromb. Haemost. 82:258-270). Active PAI-1(vitronectin-bound) inhibits proteolytic degradation of theextracellular matrix by inhibiting uPA/tPA, which in turn inhibits cellmigration and invasion (Blasi (1999) Thromb. Haemost. 82:298-304). PAI-1can exist in an active, inactive/latent or substrate-cleavedconformation (Lawrence, et al. (1997) J. Biol. Chem. 272:7676-7680;Debrock and Declerck (1998) Thromb. Haemost. 79:597-601). The PAI-1reactive center loop (RCL), located at amino acids 320-351 (Schechterand Berger (1967) Biochem. Biophys. Res. Commun. 27:157-162; Laskowskiand Kato (1980) Annu. Rev. Biochem. 49:593-626), initially interactswith uPA at Arg-346 (Lawrence, et al. (1994) J. Biol. Chem.269:27657-27662; Tucker and Gerard (1996) Eur. J. Biochem. 237:180-187)to form a stable PAI-1/uPA complex to inactivate uPA (York, et al.(1991) J. Biol. Chem. 266:8495-8500). In the active/latent configurationof PAI-1 (not bound to vitronectin), the RCL spontaneously inserts intothe β-sheet of strand 4a to stabilize the PAI-1 structure (Mottonen, etal. (1992) Nature 355:270-273; Egelund, et al. (1997) Eur. J. Biochem.248:775-785; Kjoller, et al. (1996) Eur. J. Biochem. 241:38-46). It hasbeen shown that when PAI-1 is cleaved between residues P and P′ in theRCL, PAI-1 is converted to a substrate (Lawrence, et al. (1997) supra;Debrock and Declerck (1998) supra). In the cleaved conformation, the RCLis partially inserted into β-sheet of strand A, thus making thestructure of cleaved and inactive PAI-1 more similar to each other thanto active PAI-1. However, it has been demonstrated that there aredistinct conformational differences between latent and cleaved PAI-1(Sancho (1995) Biochemistry 34:1064-1069). The PAI-1 region distant fromthe RCL contains many binding domains for regulatory molecules involvedin the proteolytic and fibrinolytic pathways. This region of PAI-1 hasinteractive sites for vitronectin (Lawrence, et al. (1994) supra;Padmanabhan and Sane (1995) Thromb. Haemost. 73:829-834; Van Meijer, etal. (1994) FEBS Lett. 352:342-346; Seiffert, et al. (1994) J. Biol.Chem. 269:2659-2666), heparin (Ehrlich, et al. (1992) J. Biol. Chem.267:11606-11611), tPA, uPA (Keijer, et al. (1991) Blood 78:401-409;Reilly and Hutzelmann (1992) J. Biol. Chem. 267:17128-17135), thrombin(Ehrlich, et al. (1992) supra), and fibrin (Ehrlich, et al. (1992)supra; Reilly and Hutzelmann (1992) supra). Through its interactionswith some of the same regulatory molecules in the proteolytic andfibrinolytic pathways, it has been demonstrated that PAI-1 (active andinactive) is also able to play a role in anti-angiogenic mechanisms(Mulligan-Kehoe, et al. (2001) J. Biol. Chem. 276:8588-8596; Schnaper,et al. (1995) J. Cell Physiol. 165:107-118; Stefansson, et al. (2001) J.Biol. Chem. 276:8135-8141).

[0007] A recent report demonstrates that when a truncated porcine PAI-1protein rPAI-1₂₃, is incubated with plasminogen and uPA, it inducesformation of an angiostatin-like protein that has proteolytic activity(Mulligan-Kehoe, et al. (2001) supra). In this reaction, angiostatin isformed from cleaved plasmin. uPA enhances the formation of theangiostatin-like protein by increasing the amount of available plasmin.The proteolytic activity of the 36 kDa angiostatin is ultimatelyinhibited by increasing amounts of rPAI-1₂₃ that are available forbinding uPA and/or plasminogen. In this second mechanism, rPAI-1₂₃reduces the numbers of uPA/plasminogen interactions; thus, reducing theamount of plasmin produced. Cultured endothelial cells exposed torPAI-1₂₃ exhibit a decrease in proliferation, increased apoptosis, anddecreased migration in the presence of VEGF. This truncated PAI-1appears to be exposing sites that participate in a functional role forPAI-1 in generating angiostatin fragments from plasmin.

[0008] Previously, zymographic analysis demonstrated the importance ofrPAI-1₂₃ interactions with uPA, plasminogen, and plasmin that result inangiostatin formation. Furthermore, it has been shown that rPAI-1₂₃blocks migration of VEGF-stimulated endothelial cells (Mulligan-Kehoe,et al. (2001) supra).

[0009] Anti-angiogenic tumor treatment strategies are based uponinhibiting the proliferation of budding vessels, generally at theperiphery of a solid tumor. These therapies are often applied to reducethe risk of micrometastasis or to inhibit further growth of a solidtumor after more conventional intervention (such as surgery orchemotherapy).

[0010] The recognition of VEGF as a primary stimulus of angiogenesis inpathological conditions has led to various attempts to block VEGFactivity. Inhibitory anti-VEGF receptor antibodies, soluble receptorconstructs, antisense strategies, RNA aptamers against VEGF and lowmolecular weight VEGF receptor tyrosine kinase (RTK) inhibitors have allbeen proposed for use in interfering with VEGF signaling (Siemeister etal. (1998) Cancer Metastasis Rev., 17(2):241-248). Monoclonal antibodiesagainst VEGF have been shown to inhibit human tumor xenograft growth andascites formation in mice (Kim, et al. (1993) Nature 362:841-844; Asano,et al. (1998) Hybridoma 17:185-90; Mesiano, et al. (1998) Am. J. Pathol.153(4):1249-1256; Luo, et al. (1998) Cancer Res. 58(12):2594-2600;Borgstrom, et al. (1996) Prostate 35(1):1-10; Borgstrom, et al. (1998)Anticancer Research 19(5B):4203-11). Moreover, U.S. Pat. No. 6,342,221to Thorpe, et al. discloses the use of anti-VEGF antibodies tospecifically inhibit VEGF binding to the VEGFR-2 receptor.

[0011] Regulation of angiogenesis by PAI proteins has also beendiscussed. U.S. Pat. No. 5,830,880 to Sedlacek, et al. discloses theexpression of PAI-1, PAI-2, PAI-3 and angiostatin through a gene therapyapproach to inhibit angiogenesis. Furthermore, RCL mutants (residues331-346) of PAI-1 have been disclosed in PCT Publication No. WO 97/39028which are resistant to elastase inactivation and/or have a high affinityfor vitronectin.

SUMMARY OF THE INVENTION

[0012] Angiogenesis is the process of blood vessel growth towards atissue in need of oxygen or an injured tissue. Angiogenesis can beeither harmful or beneficial, for example, in cases such as tumorgrowth, angiogenesis towards the tumor can supply the tumor withnutrients and support its growth, thus further harming the patient.However, in occlusive (clotting of blood vessels) diseases, the abilityto spontaneously develop collateral vessels often determines the levelof tissue viability. Thus, methods of stimulating or inhibitingangiogenic processes are needed. The present invention meets this needby providing recombinant PAI-1 proteins useful in stimulating orinhibiting angiogenesis.

[0013] One aspect of the present invention is a method of modulatingangiogenesis with a PAI-1 isoform. The method involves administering aneffective amount of a PAI-1 isoform lacking the RCL domain andcontaining a complete heparin-binding domain or lacking at least aportion of the heparin-binding domain so that angiogenesis is modulated.In one embodiment, the PAI-1 isoform lacking both the RCL domain and atleast a portion of the heparin-binding domain is useful in blocking ordecreasing angiogenesis. In a preferred embodiment, a PAI-1 isoform forblocking or decreasing angiogenesis includes rPAI-1₂₃ and rPAI-1_(Δ23).In another embodiment, the PAI-1 isoform lacking the RCL domain andcontaining a complete heparin-binding domain is useful in stimulating orincreasing angiogenesis. In a preferred embodiment, a PAI-1 isoform forincreasing or stimulating angiogenesis includes rPAI-1_(Hep23). In afurther embodiment, the PAI-1 isoform lacking the RCL domain and atleast a portion of the heparin-binding domain is useful to block therelease of VEGF from a VEGF-heparin complex thereby blockingangiogenesis. In another preferred embodiment of the present invention,the PAI-1 isoform for blocking the release of VEGF includes rPAI-1₂₃.

[0014] Other aspects of the present invention include methods ofstimulating apoptosis or reducing cell proliferation or migration. Thesemethods involve administering an effective amount of a PAI-1 isofromwhich lacks both the RCL and heparin-binding domains. In a preferredembodiment, a PAI-1 isoform for stimulating apoptosis or reducing cellproliferation or migration includes rPAI-1_(Δ23).

[0015] Another aspect of the present invention is a method of modulatingangiostatin formation. The method involves administering a PAI-1 isoformwhich lacks an RCL domain and lacks at least a portion of aheparin-binding domain so that angiostatin containing kringle 1-3 orkringle 1-4 is formed. In a preferred embodiment, a PAI-1 isoform formodulating angiostatin formation includes rPAI-1₂₃ and rPAI-1_(Δ23).

[0016] A still further aspect of the present invention is a method oftreating an angiogenesis-mediated disease. The method involvesadministering either a proangiogenic or anti-angiogenic isoform of PAI-1so that the signs or symptoms of an angiogenesis-mediated disease arereduced. In one embodiment of the present invention, an anti-angiogenicPAI-1 isoform lacks an RCL domain and lacks at least a portion of aheparin-binding domain. In a preferred embodiment, the anti-angiogenicPAI-1 isoforms include rPAI-1₂₃ and rPAI-1_(Δ23). In another embodiment,of the present invention, a proagiogenic PAI-1 isoform lacks an RCLdomain and contains a complete heparin-binding domain. In a preferredembodiment, the proangiogenic PAI-1 isoform includes rPAI-1_(Hep23).

[0017] Another aspect of the present invention is a method for producinga 34 kDa truncated plasmin proteolytic protein. The method involvescombining plasminogen and rPAI-1₂₃ for a specified amount of time andadding uPA so that a 34 kDa truncated plasmin proteolytic protein isproduced. A 34 kDa truncated plasmin proteolytic protein porduced by themethod of the invention is considered yet another aspect of the presentinvention.

[0018] A further aspect of the invention is a method for modulating theexpression of a membrane type 1-matrix metalloproteinase. The methodinvolves administering an effective amount of an plasminogen activatorinhibitor type 1 isoform lacking a reactive center loop and containing acomplete heparin-binding domain or lacking at least a portion of aheparin-binding domain so that the expression of a membrane type1-matrix metalloproteinase is modulated. When the plasminogen activatorinhibitor type isoform contains the heparin-binding domain theexpression of a membrane type 1-matrix metalloproteinase increases. Whenthe plasminogen activator inhibitor type isoform lacks a portion of theheparin-binding domain the expression of a membrane type 1-matrixmetalloproteinase decreases.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 illustrates rPAI-1 truncations relative to the human andporcine proteins. The domains that interact with specific molecules inthe proteolytic, fibrinolytic and adhesion processes are indicated witharrows and bars. Overlapping domains are indicated. Deletions were basedon the alignment of the human and porcine genes.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides methods of modulating angiogenesisthrough recombinant PAI-1 (rPAI-1) protein interactions. Four isoformsof rPAI-1 were generated, with and without the heparin-binding domainand the reactive center loop (FIG. 1, Table 1), and used to dissect thefunction of PAI-1. TABLE 1 Isoform Heparin-Binding Domain RCL DomainrPAI-1_(Hep23) Not deleted Deleted rPAI-1₂₄ Partial Deletion Not DeletedrPAI-1_(Δ23) Deleted Deleted rPAI-1₂₃ Partial Deletion Deleted

[0021] There are no known functional differences between human (huPAI-1)and porcine PAI-1 (poPAI-1) (Bijnens, et al. ((1997) Thromb. Haemost.77:350-356; Bosma, et al. (1988) J. Biol. Chem. 263:9129-9141). Two ofthe rPAI-1 proteins, rPAI-1_(Hep23) and rPAI-1_(Δ23) are encoded by DNAsequence identical to the rPAI-1₂₃ (Mulligan-Kehoe, et al. (2001) supra)except that rPAI-1_(Hep23) contains the codons for the entireheparin-binding domain, which have been completely deleted inrPAI-1_(Δ23), and 21 additional amino acid residues at the N-terminus(Met-Gln-Phe-Lys-Ile-Glu-Glu-Lys-Gly-Met-Ala-Pro-Ala-Leu-Arg-Gln-Leu-Tyr-Lys-Glu-Leu-Met-Gly-Pro-Trp-Asn-Lys;SEQ ID NO:1). The rPAI-1₂₄ was the only rPAI-1 isoform that containedthe reactive center loop (RCL) on the carboxyl terminus (amino acids320-351). Amino acid residues 262-379 in mature poPAI-1 were deletedfrom the carboxyl termini of rPAI-1₂₃, rPAI-1_(Hep23), and rPAI-1_(Δ23).The RCL was within the deleted region and, therefore, enabledexamination of the importance of a uPA site at residues 128-145, in theabsence of the primary uPA site at Arg-346. The heparin-binding domaincorresponding to amino acid residues 65-88(Lys-Ile-Glu-Glu-Lys-Gly-Met-Ala-Pro-Ala-Leu-Arg-Gln-Leu-Tyr-Lys-Glu-Leu-Met-Gly-Pro-Trp-Asn-Lys;SEQ ID NO:2) was completely deleted in the rPAI-1_(Δ23) isoform. Theheparin-binding domain of rPAI-1₂₃ lacked amino acid residues 65-82.

[0022] As will be described in greater detail, three experimentalapproaches were used to demonstrate the impact that the four rPAI-1isoforms had on angiogenic processes. The first approach was to examinethe rPAI-1 protein interactions with heparin, uPA and plasminogen. Thesecond approach explored the in vitro angiogenic activity of the rPAI-1proteins through apoptosis, cell proliferation and migrationexperiments. A third approach looked at the interactions of aVEGF-heparin complex with rPAI-1 proteins, uPA, and plasmin and theeffect on VEGF activation in rPAI-1-treated cells.

[0023] Protease activity was measured in reconstitution reactionscombining recombinant, truncated rPAI-1 molecules with uPA andplasminogen with and without heparin. Zymographic analysis indicatedthat three of the four truncated PAI-1 proteins in combination with uPAand plasminogen resulted in the production of proteolytic fragments thatmigrated at 34-38 kDa. The rPAI-1₂₃ protein induced formation of two34-38 kDa proteolytic angiostatin fragments from plasmin. However, therPAI-1_(Δ23) and rPAI-1₂₄ proteins each had a single band thatcorresponded to one of the 34-38 kDa fragments visualized in therPAI-1₂₃ products. In the case of rPAI-1_(Δ23), a proteolytic bandappeared at or near the size of the lower proteolytic fragment inducedby rPAI-1₂₃ cleavage of plasmin. The rPAI-1₂₄ protein induced aproteolytic fragment at or near the molecular mass corresponding to thelarger of the two plasmin cleavage products induced by rPAI-1₂₃. TherPAI-1_(Hep23) did not produce a proteolytic fragment at 34-38 kDa. TherPAI-1₂₃ protein (partial heparin-binding domain) maintained itsactivity when bound to heparin. Similarly, the proteolytic activityassociated with a reaction mix containing uPA, plasminogen, andrPAI-1_(Hep23) (complete heparin domain) was not altered in the presenceof heparin. The rPAI-1_(Δ23) protein (lacking a heparin-binding domain)and the rPAI-1₂₄ protein (containing the RCL and a partial heparindomain) did not demonstrate proteolytic plasmin cleavage products whenincubated with heparin. The proteolytic proteins near 80 kDacorresponded to plasmin. Proteolytic proteins near 50 kDa may representa different plasmin cleavage product containing a greater number ofplasminogen kringle domains. The function of the rPAI-1_(Hep23) isoform(complete heparin domain) in a reaction with uPA and plasminogen ascompared with rPAI-1₂₃ isoform (partial heparin-binding domain) showedthat binding to heparin did not alter the inability of rPAI-1_(Hep23) tomediate the formation of proteolytic fragments at a molecular mass near34 kDa. These experiments showed that a full heparin-binding domain mayblock the ability of PAI-1 to induce proteolytic proteins correspondingto or near the molecular mass of antiostatin containing K1-3.

[0024] In vitro analyses of angiostatin production following rPAI-1treatment zymography analysis of proteolytic angiostatin in culturemedium of rPAI-1-treated endothelial cells was conducted. The zymographyanalysis of the culture medium from rPAI-1₂₃-treated bovine aorticendothelial cells (BAEC) did not show proteolytic angiostatin at any ofthe examined time points (6, 15, 24, 36, 48, and 72 hours). On the otherhand, the medium from the rPAI-1_(Δ23)-treated cells contained asubstantial amount of proteolytic activity near 34 kDa in all of theexamined time points. Analysis of the extracellular matrix proteinsshowed that proteolytic angiostatin was produced after 72 hours ofrPAI-1₂₃ or rPAI-1_(Δ23) treatment. The molecular mass of theproteolytic fragment in rPAI-1_(Δ23)-treated cells corresponded to themolecular mass that has been shown to contain angiostatin kringles 1-3(Mulligan-Kehoe, et al. (2001) supra; Mulligan-Kehoe, et al. (1991)supra). The results indicate that rPAI-1_(Δ23) is more efficient thanrPAI-1₂₃ in producing proteolytic angiostatin in BAEC.

[0025] To further demonstrate that the plasminogen (plasmin) cleavageproducts formed in reconstitution reactions containing rPAI-1 proteins,uPA and plasminogen contained kringle domains 1-3, nitrocellulosemembranes of said cleavage products were probed with anti-kringle 1-3(angiostatin) antibodies. Samples containing 3, 15 or 30 nM of rPAI-1₂₃had fragments containing kringles 1-3 at a molecular mass correspondingto the size that was visualized by zymography. Additionally, there werefragments containing kringles 1-3 at a molecular mass near 45 kDa thatcorrespond to the reported size of kringles 1-4, and 70-80 kDa thatcorrespond to the size of plasmin. The reaction product of 15 nM ofrPAI-1_(Δ23), uPA and plasminogen contained angiostatin kringles 1-3 at34-36 kDa. However, that fragment was absent when 3 or 30 nM ofrPAI-1_(Δ23) were part of the reaction with uPA and plasminogen. In thereactions containing either rPAI-1₂₄ or rPAI-1_(Hep23), plasminogenkringles 1-3 were not present at 34-36 kDa. There were detectablefragments containing kringles 1-3 near 45 kDa in the reactionscontaining 3 nM of rPAI-124. These data showed that the reaction of uPAand plasminogen with all concentrations of rPAI-1₂₃ resulted in theformation of plasminogen kringles 1-3 (angiostatin) at 34-36 kDa, whichis consistent with the molecular mass of the proteolytic fragmentsobserved on the zymogram. Additionally, there is a greater amount ofkringles 1-3 at 45 kDa which is representative of a less potentangiostatin.

[0026] Immunoblot analyses of angiostatin in culture medium ofrPAI-1-treated endothelial cells was also conducted. The results of theimmunoblot analysis of the culture medium protein isolated from rPAI-1treated or untreated BAEC showed that, at all three time points (24, 36,and 48 hours) and in all three test samples (no treatment,rPAI-1₂₃-treated, and rPAI-1_(Δ23)-treated), there were predominantproteins containing angiostatin kringles 1-3 at a molecular weightbetween 34 and 50 kDa and a less predominant fragment at 28 kDa. Thekringles 1-3 fragments near 50 kDa were less intense in the rPAI-1_(Δ23)samples at 24 hours, but they became more intense at each later timepoint. The rPAI-1_(Δ23) samples at 36 and 48 hours displayed apronounced K1-3 fragment between 28 and 34 kDa. In the 36 and 48 hourrPAI-1₂₃-treated cells, there was an additional processed/cleavedkringles 1-3 fragment beneath the fragment observed between 34 and 50kDa. The 36 and 48 hour untreated samples did not contain additionalcleavage products. The data from these experiments demonstrate that inrPAI-1₂₃-treated cells at 36 and 48 hours, the fragments containingkringles 1-3 have undergone additional processing to result inangiostatin of a lower molecular mass; a size of angiostatin withgreater anti-angiogenic activity (O'Reilly, et al. (1994) supra; Lucas,et al. (2000) Biochemistry 39:508-515; Cao, et al. (1999) supra; andO'Reilly, et al. (1996) supra). The amount of angiostatin is clearly ingreater abundance in rPAI-1_(Δ23)-treated cells.

[0027] The results provided herein indicated that rPAI-1₂₃ maypreferentially cleave plasminogen into angiostatin while rPAI-1_(Δ23)may cleave plasmin into angiostatin with associated proteolyticactivity. Thus, further analysis of angiostatin produced in biochemicalreactions containing rPAI-1 proteins was performed wherein angiostatinwas produced in varied permutations with rPAI-1 and uPA. This analysisprovided a more extensive examination of the plasminogen/plasmincleavage products in reactions containing either rPAI-1₂₃ orrPAI-1_(Δ23) and two-chain uPA (tcuPA). Immunoblots, containing theproducts of the biochemical reactions, probed for angiostatin kringles1-3, kringle 4, and mini-plasminogen were examined. In these reactions,the rPAI-1 protein was first reacted with 0.25 IU of tcuPA before adding1 IU of plasminogen. The rPAI-1₂₃ samples contained kringles 1-3 (K1-3)at 28-34 kDa, 45 kDa, 50 kDa, and near 70 kDa. The intensity of the 34kDa fragment increased with increasing concentrations of rPAI-1₂₃. Atthe highest concentration of rPAI-1₂₃ (30 nM), the 45 kDa K1-3 fragmentwas reduced and there was also a decrease in the 70 kDa fragment. Inreactions containing rPAI-1_(Δ23), the 24-28 kDa K1-3 fragments wereonly present when rPAI-1_(Δ23) was at 15 nM. There were changes in theintensity of the fragments containing K1-3 at 45 and 50 kDa, which alsoappeared to be shifting upward. The intensity of plasmin at 70 kDa wasnot reduced in samples where the K1-3 fragments were increased.

[0028] When the same reactions were probed for kringle 4 (K4), there wasa scant amount of K4 near 50 kDa in the reactions containing higherconcentrations of rPAI-1₂₃ (15 and 30 nM)) and rPAI-1_(Δ23). Thegreatest intensity was in the reaction which contained 15 nM ofrPAI-1_(Δ23). In the rPAI-1_(Δ23) samples, there were two fragments near45-50 kDa. There was not any detectable plasmin or plasminogen at 70-80kDa in the reactions containing rPAI-1₂₃, but they were detect-able inthe rPAI-1_(Δ23) samples. A mini-plasminogen (MP) probe of the reactionmixtures did not detect any fragments below 70 kDa and MP was onlyvisible at levels slightly above background near 70 kDa.

[0029] Reaction mixtures were then probed for K1-3, K4, and MP in apermutation where uPA, plasminogen, and either rPAI-1₂₃ or rPAI-1Δ23were added simultaneously. In the K1-3-probed reactions containingrPAI-1₂₃, the K1-3 fragments at 28-34 kDa were nearly undetectable, the45 and 50 kDa fragments in the samples containing 3 and 15 nM ofrPAI-1₂₃ were intense, and intense K1-3 protein at 70-80 kDa was presentat all concentrations of rPAI-1₂₃ (3, 15, and 30 nM). The reactionsrPAI-1_(Δ23) showed a dramatic difference in K1-3 fragments as comparedto the rPAI-1₂₃ samples. The rPAI-1_(Δ23) samples all displayed adistinct 28-34 kDa fragment. A less intense doublet near 45 kDa wasvisualized in samples containing 3 and 15 nM of rPAI-1_(Δ23). The 50 and70-80 kDa fragments were absent at all three concentrations ofrPAI-1_(Δ23) (3, 15, and 30 nM).

[0030] A K4 probe of the same reaction permutation containing rPAI-1₂₃revealed the presence of angiostatin fragments near 45 kDa at all threeconcentrations of rPAI-1₂₃; a slight angiostatin fragment at 50 kDa inreactions containing 3 and 15 nM of rPAI-1₂₃; and K4 was not detectablenear 70-80 kDa when rPAI-1₂₃ was present at 15 and 30 nM. Amini-plasminogen probe of the reaction mixture showed a fragment at 34kDa when rPAI-1₂₃ was present at 15 and 30 nM. The MP fragment was of aslightly smaller molecular mass than the K4 probed fragments. The samplereaction mixtures containing rPAI-1_(Δ23) did not reveal any detectableK4 or mini-plasminogen in this permutation.

[0031] The immunoblots indicated that there were differences inangiostatin produced in reactions with rPAI-1₂₃ and rPAI-1_(Δ23). Thedifferences were not only due to structural differences in rPAI-1, butwere, also in part, due to molecular interactions that seeminglyoccurred in response to exposed domains in rPAI-1₂₃ and rPAI-1_(Δ23).The alteration in angiostatin fragments containing K1-3 indicated thatat high concentrations of rPAI-1_(Δ23), which exceeded a 1:1 molar ratiowith uPA, plasminogen was cleaved into angiostatin fragments near 34kDa. However, in the permutation where all three molecules were allowedto interact simultaneously, the cleavage product was angiostatincontaining K1-4 near 45 kDa. When rPAI-1₂₃ was in excess, then the K1-3fragments at 50 and 45 kDa diminished in intensity. These data show thata fragment containing kringles 1-4 was the preferred cleavage product ina reaction where uPA first reacted with rPAI-1_(Δ23) before plasminogenwas added. When rPAI-1_(Δ23), uPA and plasminogen were simultaneouslyadded to a reaction, two sets of angiostatin fragments containing K1-3were produced. While it appeared that the 34 kDa fragment was moreabundant, there may have been two cleavage products at the onset or theK1-4 product may have underwent additional cleavage/processing to 34 kDaas rPAI-1_(Δ23) concentrations were was increased. The significantdifference in these reactions, when compared to the permutation whereuPA was first reacted with rPAI-1_(Δ23) before adding plasminogen, wasthe complete loss of plasmin and/or plasminogen. These collective dataindicate that both rPAI-1₂₃ and rPAI-1_(Δ23) may bind uPA and/orplasminogen and the varied interactions alter the cleavage site inplasminogen and/or plasmin.

[0032] As the data indicated that rPAI-1₂₃ and rPAI-1_(Δ23) may cleaveplasminogen, additional biochemical reactions were performed to analyzeinteractions of rPAI-1 with plasminogen. Increasing concentrations ofrPAI-1₂₃ and rPAI-1_(Δ23) were each incubated with plasminogen (withoutuPA) and then immunoblots containing each reaction mixture were probedfor mini-plasminogen. The probed immunoblots revealed a distinctplasminogen cleavage product near 50 kDa in all reactions containing therPAI-1₂₃ protein (3, 15 and 30 nM). In the same reactions, acorresponding decrease in plasminogen at 80 kDa was seen as well,clearly demonstrating that plasminogen was cleaved into the smallerproduct. In contrast, the rPAI-1_(Δ23) protein did not cleaveplasminogen, even when increasing concentrations of the recombinantprotein were added. Based on the mini-plasminogen antibody recognition,the 50 kDa cleavage product contained either multiple kringle domains toinclude K5 or a combination of kringle domains and the serine proteasedomain. The data demonstrates that the rPAI-1₂₃ recombinant proteincleaves plasminogen, while the rPAI-1_(Δ23) protein does not.

[0033] To evaluate which cleavage products in reactions containingrPAI-1₂₃ resulted from plasminogen vs. plasmin, the cleavage products ina reaction containing rPAI-1₂₃ and plasminogen were examined.Additionally, the effect that uPA may potentially have on rPAI-1₂₃cleavage of plasminogen was examined. These experiments showed distinctrPAI-1-cleaved plasminogen fragments containing K1-3 at 50 kDa, between28-34 kDa, and predominantly at 45 kDa. These data demonstrate that the28-34 kDa angiostatin was K1-3 cleaved from plasminogen. As increasingconcentrations of uPA were added to the rPAI-1₂₃ plasminogen complex,the K1-3 at 28-34 kDa diminished and was eventually undetectable. Thisshowed that uPA was able to compete rPAI-1₂₃ away from plasminogen in auPA dose-dependent manner. A decrease in the amount of plasmin at 72 kDawas seen as compared to the plasmin formed in a uPA and plasminogenreaction. The results of these experiments indicated that rPAI-1₂₃cleaved plasminogen into angiostatin K1-3, a more potent angiostatinfragment (Cao, et al. (1996) supra); uPA could reverse that cleavage bycompeting rPAI-1₂₃ away from plasminogen; and rPAI-1_(Δ23) does notcleave plasminogen. These results further indicate that the rPAI-1₂₃protein has both binding capabilities which have the potential toultimately regulate plasmin formation.

[0034] Results provided herein demonstrated rPAI-1_(Δ23) did not cleaveplasminogen, however the effect that uPA may have on the formation ofangiostatin was not investigated in reactions containing rPAI-1_(Δ23)and plasminogen. Therefore, analysis of angiostatin in biochemicalreactions containing rPAI-1_(Δ23), uPA and plasminogen was performed. AsK4 and MP were not identified in reactions where rPAI-1_(Δ23), uPA, andplasminogen were added simultaneously to a reaction, concentrations ofplasminogen were increased to 2 IU in the next series of experimentswhere uPA and rPAI-1_(Δ23) concentrations were varied. Biochemicalreactions, containing rPAI-1_(Δ23), uPA and plasminogen, were performedto identify differences in angiostatin production that may occur as aresult of the order in which the molecules were added to the reactionmixture.

[0035] Immunoblots of products of a reaction where all three reactantswere added simultaneously, then incubated at 37° C. for two hours showedtwo distinct subsets of proteins containing K1-3. One subset had amolecular mass of approximately 45 kDa, the size reported forangiostatin K1-4 (O'Reilly, et al. (1994) supra, Cao, et al. (1996)supra). The other subset of protein fragments had a molecular massbetween 28 and 36 kDa, the size reported for angiostatin K1-3 (O'Reilly,et al. (1994) supra, Cao, et al. (1996) supra). The most observabledifferences in the two subsets of fragments containing K1-3 were thefollowing. 1) The intensity of the 28-36 kDa protein fragments wasgreater than the 45 kDa fragments. 2) The intensity of the fragmentsnear 45 kDa decreased as the concentration of rPAI-1_(Δ23) wasincreased; as the uPA concentration was increased, the intensity of the45 kDa fragments increased, but not to the level observed whenrPAI-1_(Δ23) was at the lowest concentration (3 nM). 3) As theconcentration of rPAI-1_(Δ23) was increased, there was a shift in themolecular mass of both subsets of fragments containing K1-3; the shiftwas greater when uPA was at lower concentrations, which indicated thatan increase in uPA stabilized the reaction and the site ofplasminogen/plasmin cleavage. Alternatively, uPA may be in sufficientquantity to eliminate competition between rPAI-1_(Δ23) and plasminogenfor uPA binding sites. 4) There were differences in the amount ofplasmin or plasminogen that remained uncleaved when rPAI-1_(Δ23) waspart of the reaction. In reactions where uPA was at the highestconcentration (0.25 Units), plasmin and plasminogen were not detectable.When uPA was reduced by one-fourth, greater amounts of plasmin andplasminogen remained uncleaved. These observations indicated that uPAwas bound to rPAI-1_(Δ23) when the rPAI-1_(Δ23) was in excess, such thatuPA was unavailable to cleave plasminogen to form plasmin.

[0036] Further analysis of the interaction of tcuPA with rPAI-1_(Δ23)was conducted to determine if this interaction stabilized the cleavageof plasmin or plasminogen into angiostatin. The results of immunoblotsof biochemical reactions in which rPAI-1_(Δ23) and uPA were firstreacted before adding plasminogen showed stabilized angiostatinfragments in two distinct subsets of fragments: one subset near 50 kDaand the other subset at 34 kDa. The intensity of the fragment near 50kDa decreased with increasing amounts of rPAI-1_(Δ23). The plasminremaining in the reaction mixtures was undetectable at the higherconcentrations of rPAI-1_(Δ23), independent of the uPA concentration.Differences between the results of these experiments and those providedabove were that the molecular mass of the angiostatin fragments did notshift when rPAI-1_(Δ23) and uPA were reacted first before addingplasminogen, which indicated that the interaction between uPA andrPAI-1_(Δ23) stabilized the cleavage of plasmin.

[0037] Results provided herein indicate a competition betweenrPAI-1_(Δ23) and plasminogen for binding uPA to result in a fraction ofuncleaved plasmin or plasminogen. Thus, plasmin was made in a reactioncontaining uPA and plasminogen before adding rPAI-1_(Δ23). In thesereactions, the two subsets of angiostatin fragments were produced. Theangiostatin fragments shifted slightly upward when the concentration ofrPAI-1_(Δ23) was increased. There was a diminution in both subsets ofthe angiostatin fragments when 30 nM of rPAI-1_(Δ23) was in the reactionmix. The proteins corresponding to the molecular mass of plasmin wereabsent when low concentrations of uPA were combined with middleconcentrations of rPAI-1_(Δ23) (15 nM) and when both uPA andrPAI-1_(Δ23) were at their highest concentrations (0.25 Units and 30 nM,respectively). There was a greater intensity in plasminogen when thereactions contained the highest concentration rPAI-1_(Δ23) (30 nM) andthe low and mid concentrations of uPA (0.025 and 0.05 Units). Those samereactions also contained less angiostatin at 45 and 28-36 kDa, whichindicated that: uPA conversion of plasminogen into plasmin was notexhausted after 1 hour, and that rPAI-1_(Δ23) was unable to cleave theresidual plasminogen; increasing concentrations of rPAI-1_(Δ23) wereable to compete with plasminogen for binding to uPA to result in areduction in plasmin levels; or rPAI-1_(Δ23) can bind a uPA/plasminogencomplex, alter the conformation of uPA or plasminogen to result ininefficient conversion of plasminogen into plasmin or a reduction inepitope binding by the antibody.

[0038] The reaction mixtures, containing rPAI-1_(Δ23), uPA andplasminogen reacted in varied permutations, were also probed forplasminogen K4. K4 was present at 45 kDa in all reactions where uPA wasfirst reacted with rPAI-1_(Δ23) before adding plasminogen. The intensityof the fragments at 45 kDa decreased when the rPAI-1_(Δ23) concentrationwas increased and uPA was at low and mid concentrations. At the highestconcentration of uPA (0.25 Units), the intensity of the fragmentscontaining K4 declined at all concentrations of rPAI-1_(Δ23) (3, 15, and30 nM). Additionally, when plasmin was first made (uPA+plasminogen)before adding rPAI-1_(Δ23), K4 was detectable at the 45 kDa range whenrPAI-1_(Δ23) was at low and mid concentrations (3 and 15 nM), but wasabsent when rPAI-1_(Δ23) was at the high concentration (30 nM). Therewas a slight shift in the angiostatin K1-4 fragment as the uPAconcentration was increased. When all three reactants weresimultaneously added to the reaction mixture and the resulting reactionproducts were probed for plasminogen K4, there was very little evidenceof kringle 4. It was slightly detectable at 45 kDa when rPAI-1_(Δ23) wasat the lowest concentration, but was not detectable at 28-38 kDa. Thesedata indicated that the binding domain for K4 was unavailable in most ofthe reaction mixtures.

[0039] None of the reactions containing rPAI-1_(Δ23) showed K4 at themolecular mass corresponding to plasminogen or plasmin. The subset ofangiostatin fragments at 28-36 kDa was not present when probed for K4thus providing verification that the 28-36 kDa was K1-3 and not K1-4.There was no evidence of K1-4 in the reaction mixtures where uPA andplasminogen were first reacted before 30 nM of rPAI-1_(Δ23) was added.In these same samples, K1-3 were diminished at 45 kDa and 28-34 kDa.These combined data indicate that there was a conformational change inplasminogen and/or plasmin in both permutations, which made the epitopesfor K1-3 and K4 less available and unavailable, respectively.

[0040] Plasminogen fragments were undetectable on immunoblots,containing plasminogen, uPA and rPAI-1_(Δ23) reaction mixtures in allpermutations, which were probed for mini-plasmin (K5+the serine proteasedomain).

[0041] Collectively these data demonstrate that in reactions with uPAand plasminogen, the rPAI-1_(Δ23) protein is responsible for thecleavage of plasmin into angiostatin K1-3 at 28-36 kDa and angiostatinK1-4 at 45 kDa. Plasminogen remained in the reactions containing 30 nMof rPAI-1_(Δ23); thus validating the results which showed thatrPAI-1_(Δ23) was unable to cleave plasminogen. Increasing concentrationsof uPA along with increasing concentrations of rPAI-1_(Δ23) appear toreduce uPA interactions with plasminogen, which was further evidenced bya decrease in plasmin cleavage products. The order in which thereactants were added appeared to alter the cleavage site in plasmin orplasminogen. In the biochemical reactions, an rPAI-1_(Δ23)-uPA complexstabilized the cleavage of plasmin into angiostatin K1-3 and K1-4.

[0042] The collective data indicated that tcuPA binds rPAI-1_(Δ23).Therefore, experiments were performed to examine the interactions ofrPAI-1₂₃ and rPAI-1_(Δ23) with scuPA and tcuPA. Single-chain uPA wasable to undergo a small degree of autocleavage, which was visible in thecontrol scuPA as a fraction of tcuPA near 34 kDa. The data showed thatwhen either rPAI-1₂₃ or rPAI-1_(Δ23) were reacted with scuPA, there wasa decrease in the amount of detectable scuPA and tcuPA as each rPAI-1protein concentration was increased. These results indicated that bothrPAI-1₂₃ and rPAI-1_(Δ23) bind scuPA. As the concentration of rPAI-1 wasincreased, the binding interactions with uPA altered the conformation ofuPA such that the epitope was not recognized by the antibody. When 30 nMof rPAI-1_(Δ23) were reacted with scuPA, there was a substantial upwardshift in the molecular mass of scuPA. Since the reacted proteins wereelectrophoresed on an SDS, non-reducing polyacrylamide gel, these dataindicate that a covalent bond was formed between the two proteins.

[0043] The rPAI-1 proteins were also reacted with tcuPA and the reactionmix was probed for uPA. There was a slight upward shift in the molecularmass of tcuPA that had been reacted with plasminogen, 3 nM rPAI-1₂₃, and3 nM rPAI-1_(Δ23). There was a dramatic shift in tcuPA (from near 34 kDato greater than 52 kDa) when it was reacted with 15 and 30 nM ofrPAI-1₂₃. There was also a significant, but less consistent upward shiftin the molecular mass of tcuPA reacted with 15 and 30 nM ofrPAI-1_(Δ23). The shift in the molecular mass of tcuPA indicated thatboth rPAI-1₂₃ and rPAI-1_(Δ23) bound tcuPA and that a covalent bond wasformed in the binding interactions to account for the obvious shift inmass.

[0044] Angiostatin production was examined when rPAI-1_(Δ23) andrPAI-1₂₃ were added simultaneously to a reaction mixture withplasminogen and either scuPA or tcuPA. Immunoblots containing theproducts of the reactions were probed for angiostatin K1-3. When scuPA,rPAI-1₂₃, and plasminogen were added simultaneously to a reactionmixture, K1-3 were present as two distinct fragments between 34 and 50kDa and less pronounced near 28 kDa. It appeared that the cleavageproduct was predominantly from plasmin. There was a slight, subtleupward shift in the molecular mass of the angiostatin fragments producedin a reaction with rPAI-1₂₃, tcuPA, and plasminogen. In these reactions,the angiostatin fragments were predominantly plasmin cleavage productsas evidenced by the depletion in plasmin near 70 kDa. Alternatively,rPAI-1₂₃ may preferentially cleave plasminogen and/or bind uPA toinhibit plasmin formation altogether.

[0045] When rPAI-1_(Δ23) was incubated with tcuPA and plasminogen,angiostatin fragments were observed near 45 kDa and between 28-34 kDa;plasmin and plasminogen were undetectable at 70-80 kDa. As theconcentration of rPAI-1_(Δ23) was increased, there was a reduction inthe angiostatin fragments at 45 kDa. However, when scuPA was substitutedfor tcuPA, the same angiostatin fragments were present, but they wereshifted upward with increasing concentrations of rPAI-1_(Δ23). WhenrPAI-1_(Δ23) was present at 30 nM, the kringle fragments were notpresent and plasminogen shifted to a molecular mass that exceeded theplasminogen control. Thus, uPA may have preferentially interacted withplasminogen to produce plasmin, which was then completely cleaved byrPAI-1_(Δ23) or the complex that was formed with 30 nM of rPAI-1_(Δ23)prevented plasmin formation and subsequent cleavage. This was evidencedbiochemically by a shift in the molecular mass of plasminogen. Theseresults demonstrate that both tcuPA and scuPA have the potential tosubstantially effect rPAI-1_(Δ23)-induced cleavage of plasmin intoangiostatin. However, differences in the uPA conformation, that wasscuPA versus tcuPA, seemingly had an effect on angiostatin processing(additional cleavage) in reactions containing rPAI-1₂₃. Nevertheless, inall cases, plasmin levels were depleted.

[0046] To further analyze the nature of the 34 kDa proteolyticplasminogen product identified herein, biochemical experiments wereconducted to analyze the plasminogen cleavage kinetics in reactionscontaining rPAI-123 and plasminogen. An rPAI-1₂₃ and plsaminogenreaction mixture was equally distributed to multiple tubes so that uPAcould be added at 15, 30, 45 and 60 minutes after the rPAI-1₂₃ andplasminogen reaction was initiated at 37° C. The reation was stopped 2hours after initiation of the incubation of the first samples(plasminogen and rPAI-1₂₃+plasminogen). Accordingly, the formation ofthe truncated proteolytic plasminogen product near 34 kDa and thecleavage of plasminogen by rPAI-1₂₃ was observed. Proteolytic proteinsproduced in the reations were evaluated by zymography on a 15% SDSnon-reducing polyacrylamide gel containing 1.3% casein. The reactionmixtures were electrophoresed on 4-20% gradient SDS, non-denaturingpolyacrylamide gels. The transferred proteins were probed with anantibody specific for mini-plasmin (kringle 5+the serine proteasedomain) to analyze plasminogen cleavage products that contained theprotease domain.

[0047] Zymogram analysis clearly demonstrated that two proteolyticfragments were present near 34 kDa only in reactions where uPA was addedto reaction mixtures containing plasminogen and rPAI-1₂₃. In these samereactions, there were additional proteolytic fragments with lessactivity near 50 kDa and 80 kDa (the molecular mass of intact plasmin).The amount of intact plasmin increased when uPA was added to thereaction. Concurrently, proteolytic proteins at 50 and 34 kDa increasedas well. The intensity of proteolytic activity near 34 kDa was muchgreater when uPA was reacted with the rPAI-1₂₃ and plasminogen reactionmixture for 45 minutes as compared to shorter times with uPA in themixture. In plasminogen and uPA reactions, in the absence of rPAI-1₂₃,intact plasmin near 80 kDa was not apparent until 60 minutes ofincubation at 37° C.

[0048] The same reaction mixtures were analyzed for plasminogen miniplasmin by immunoblot analysis. Plasminogen cleavage products bindingthe anti-mini-plasmin antibody were identified near 50 kDa and 34 kDa,corresponding in molecular mass to the proteolytic proteins produced inthe same reactions. There was no significant variability in fragmentintensity in reactions that contained rPAI-1₂₃ and plsminogen orrPAI-1₂₃ and plasminogen and uPA. The amount of plasminogen at 80 kDawas substantially greater than the amount of the cleavage product. Verylittle of the plasminogen was converted to intact plasmin when relativeintensities of all fragments were compared between immunoblots andzymograms. These results indicate that uPA plays a role in the reactionmixture by converting intact plasminogen into intact plasmin andconverting cleaved plasminogen into a truncated plasmin of 34 kDa.rPAI-1₂₃-cleaved plasminogen was essential for the formation of thetruncated plasmin and seemingly competed with uPA in a standardplasminogen reaction. The 34 kDa proteolytic protein that corresponds torPAI-1₂₃-induced cleaved plasminogen product appears to bephysiologically relevant as it is found in commercially available,purified plasmin and atherosclerotic plaques.

[0049] Accordingly, another aspect of the present invention is method ofproducing a 34 kDa truncated plasmin proteolytic protein. The truncatedplasmin protein may be generated by combining plasminogen and rPAI-1₂₃for a specified amount of time and subsequently adding uPA to thereaction. It is contemplated that one may recognize the production of a34 kDa truncated plasmin proteolytic protein by its activity, size, andbinding by an antibody specific for mini-plasmin. Preferably, the timerequired for rPAI-1₂₃ to enhance the conversion of plasminogen toplasmin is 15, 30, 45, 60 minutes or more, most preferably more than 45minutes. The time required to convert plasmin to truncated plasmin viauPA will be dependent on the desired amount of trucated plasmin to beproduced, however, times of 15, 30, 45, 60 minutes or more, preferablymore than 45 minutes, are contemplated. The amount of reactants combinedin accordance with this method of the invention will be dependent on theamount of product desired and may vary with the incubation time (e.g.,longer incubation times may be necessary when lower concentrations ofrPAI-1₂₃ are used to enhance the conversion of plasminogen to plasmin).It is contemplated that a 34 kDa truncated plasmin proteolytic proteinproduced by this method of the invention may be useful as ananti-angiogenic agent.

[0050] Reduced plasmin levels attributed to rPAI-1₂₃ indicated thatthere would possibly be a reduction in downstream activitation ofplasmin by matrix metalloproteinases (MMPs) An RNase protection assaywas performed by probing RNA isolated from endothelial cells treatedwith rPAI-1₂₃, rPAI-1_(Hep23) or VEGF with a set of probes from variousMMPs. It was found that at 18 hours of treatment, pro-angiogenicrPAI-1_(Hep23) and anti-angiogenic rPAI-1₂₃ displayed significantdifferences in membrane type 1-matrix metalloproteinase (MT1-MMP)transcripts. The rPAI-1₂₃-treated cells had more than a 400% reductionin MT1-MMP transcript, as compared to rPAI-1_(Hep23), and a 360%reduction when compared to VEGF-stimulated transcript. MT1-MMP has beenshown to up-regulate VEGF expression (Sounni, et al. (2002) FASEB J.16(6):555-64; Deriyugina, et al. (2002) Cancer Res. 62(2):580-8). Thus,results provided herein indicate that the rPAI-1₂₃ down-regulation ofMT1-MMP and concurrent down-regulation of VEGF may, in part, account fora more profound anti-angiogenic activity of rPAI-1₂₃.

[0051] Biochemical and in vitro analysis of angiostatin formationindicated that rPAI-1₂₃ and rPAI-1_(Δ23) proteins selectively modulatedangiostatin formation. Accordingly, in one embodiment of the presentinvention, rPAI-1 proteins lacking a reactive center loop and lacking atleast a portion of a heparin-binding domain may be used in a method formodulating the formation of angiostatin which contains kringles 1-3 orkringles 1-4. In a preferred embodiment, a rPAI-1 protein for use inmodulating angiostatin formation is rPAI-1₂₃ or rPAI-1_(Δ23).

[0052] In vitro anti-angiogenic activity of the rPAI-1 proteins wasanalyzed. In an Annexin V binding assay, it was demonstrated thatadherent bovine aortic endothelial cells (BAEC), 39% of the rPAI-1₂₃ and19% of rPAI-1_(Δ23)-treated cells, were undergoing apoptosis. Whereas,only 4-7% of the rPAI-1_(Hep23), rPAI-1₂₄, and yeast-treated cells wereapoptotic, a value comparable to the endothelial control. This indicatedthat heparin-binding to rPAI-1_(Hep23) protects the endothelial cellsfrom apoptosis. Therefore, proteolytic cleavage products of rPAI-1₂₃ andrPAI-1_(Δ23) may be involved with the induction of apoptosis. Thus, inanother embodiment of the present invention, rPAI-1 proteins lacking areactive center loop and lacking at least a portion of a heparin-bindingdomain may be used in a method for stimulating apoptosis. In a preferredembodiment, a rPAI-1 protein for use in stimulating apoptosis isrPAI-1₂₃ or rPAI-1_(Δ23).

[0053] To further investigate the in vitro anti-angiogenic activity ofthe rPAI-1 proteins, cell proliferation in the presence of rPAI-1proteins was examined. Bovine aortic endothelial cells were evaluatedfor their ability to proliferate after exposure to rPAI-1 proteins. Thecontrol cells proliferated at a rate of approximately one doubling in 48hour. The rPAI-1₂₄-treated cells did not double in number during thefirst 48 hours, but doubled in the subsequent 48 hours. The rPAI-1₂₃—and rPAI-1Δ23-treated cells did not increase in number in the 96 hourtest period. In fact, the rPAI-1₂₃-treated cells decreased their numberby 80% between 48 and 96 hours. There were 46% fewer apoptoticrPAI-1_(Δ23)-treated cells. Nearly 100% of the rPAI-123- andrPAI-1_(Δ23)-treated endothelial cells examined in a BrdU labeling assayincorporated BrdU into DNA; thus supporting low density in rPAI-1₂₃ andrPAI-1_(Δ23)-treated cells as a result of reduced number, and not lossof proliferation capability. The heparin sulfate-binding rPAI-1 proteinsupports full proliferation and cell survival while the poor ornon-heparin sulfate-binding proteins impair cell number. Accordingly, inanother embodiment of the present invention rPAI-1 proteins lacking areactive center loop and lacking at least a portion of a heparin-bindingdomain may be used in a method for effectively reducing or inhibitingcell proliferation. In a preferred embodiment, a rPAI-1 protein for usein reducing or inhibiting cell proliferation is rPAI-1₂₃ orrPAI-1_(Δ23).

[0054] Experiments focusing on tubule formation were also conducted toexplore the in vitro anti-angiogenic activity of the rPAI-1 proteins.Chick aortic arch rings from 14-day chick embryos formed tubules inMATRIGEL™. The tubules proliferated and migrated extensively whenstimulated with bovine brain extract (BBE). The rings that were exposedto rPAI-1_(Hep23)-BBE had a proliferation and migration rate at leastequivalent to the control. In contrast, after 3 days of exposure torPAI-1₂₃-BBE, new tubules extended from the aortic rings toapproximately 50% of the length measured in the control orrPAI-1_(Hep23)-treated rings. By day 4, that difference was 65%. Thenewly formed tubules from the aortic rings treated with rPAI-1_(Δ23)migrated about 50% less than the control on day 3. That differenceremained nearly the same on day 4. There was a greater amount ofproliferation near the periphery of the ring of rPAI-1_(Δ23)-treatedsamples when compared to the rPAI-1₂₃-treated rings. The branches of therPAI-1₂₃— and rPAI-1_(Δ23)-treated rings were more flattened and tightlyconnected. Their branches appeared to fuse as they extended parallel.The differences observed in the newly formed tubules in therPAI-1-treated samples are consistent with the data presented above,where it was shown that unlike rPAI-1₂₃ and rPAI-1_(Δ23), therPAI-1_(Hep23) does not induce the formation of proteolytic fragments ata 34-38 kDa molecular mass corresponding to plasminogen kringles 1-3 anddoes not induce apoptosis in BAEC with a concomitant reduction in cellnumber.

[0055] The aortic ring studies indicated that rPAI-1_(Hep23) did notblock migration or proliferation of sprouting tubules in MATRIGEL™.Therefore, the ability of rPAI-1_(Hep23) to stimulate tubule migrationand proliferation in the absence of angiogenic growth factors, bFGF andVEGF was examined in an ex vivo assay. Aortic rings stimulated with acombination of VEGF and bFGF were used as a positive control. Theseexperiments showed that rPAI-1_(Hep23) was able to stimulate angiogenictubules at a level comparable to that observed and measured in embryonicaortic rings stimulated with combined VEGF and bFGF.

[0056] An evaluation of rPAI-1₂₃ inhibition of VEGF in a breast tumorwas performed ex vivo in excised murine breast adenocarcinoma tumors.The tumors were cut into equivalent, spoke-like pieces from the centerof the excised tumor. The tumor pieces were placed in 6-well cellculture plates coated with MATRIGEL™. The tumor pieces were then coatedwith an additional thin layer of MATRIGEL™, incubated at 37° C. in humanendothelial serum-free medium containing 10% FBS, VEGF (10 ng/mL) orVEGF (10 ng/mL) and rPAI-1₂₃ (0.6 nM). Tumors were cultured for 7 daysduring which time tubes formed in MATRIGEL™. Tumor cells metastasizedand migrated to the extending vessels indicating that VEGF had causedthe vessels to become permeable and hence releasing chemotactic factors.Conversely, rPAI-1₂₃/VEGF-treated tumors formed fewer numbers of tubes,appeared to be undergoing apoptosis, and were not proliferating. Thus,these data show in vivo inhibition of VEGF function in a tumor. In vivoanalysis of the proangiogenic properties of rPAI-1_(Hep23) were alsoconducted. The cumulative results from three experiments indicated thatproangiogenic rPAI-1_(Hep23) protein stimulated 163% more neoangiogenicvessels than the MATRIGEL™ pellets containing VEGF/bFGF. The rPAI-123protein, which has anti-angiogenic properties, reduced the number ofneoangiogenic vessels to 1.3% of those formed in growthfactor-stimulated pellets. The results indicate that rPAI-1_(Hep23) hasproangiogenic properties which are growth factor independent whilerPAI-1₂₃ is anti-angiogenic.

[0057] The biochemical data provided herein demonstrated formation of auPA•rPAI-1 complex that could possibly alter the activity of uPA or therPAI-1 proteins. Therefore, the ability of endothelial cells to formtubes, migrate/extend, and proliferate in MATRIGEL™ following 24 hoursof treatment with: each rPAI-1 protein; uPA and rPAI-1 addedsimultaneously, but independently; or a uPA•rPAI-1 complex wereexamined. Each rPAI-1 protein concentration was reduced so that uPA wasin excess. It was determined that BAEC treated with rPAI-1_(Δ23) formeda greater number of migrating tubules in MATRIGEL™ when compared tountreated or rPAI-1₂₃-treated BAEC. Further, connecting, migratingtubules and enhanced proliferation at focal centers were the obviouscharacteristics that resulted from scuPA and tcuPA treatment. TherPAI-1_(Δ23)-treated cells had fewer branch points and tubule extensionsthan scuPA- and tcuPA-treated cells. The rPAI-1_(Δ23)-treatedendothelial cells that migrated in MATRIGEL™ formed tubules thatappeared to be proliferating as they extend to result in greater tubulewidth; this effect was also seen in tcuPA-treated cells. Moreover, theeffects of an rPAI-1_(Δ23)•tcuPA complex added to the BAEC increasedproliferation at the focal centers, and increased tubule migration andextension. However, there was no apparent sprouting from the migratingtubules and the tubules appeared to be thinner. The overall effect wasincreased proliferation, migration and connecting of tubules, but areduction in tubule width. Furthermore, when rPAI-1_(Δ23) and tcuPA wereadded separately, but simultaneously, to the cultured cells, the tubuleextension between focal centers was shortened. There was moreproliferation along the migrating tubules, but the overall tubuledensity was reduced when compared with the tcuPA-treated cells. Inaddition, BAEC treated with a rPAI-1_(Δ23)•scuPA reaction mixturedisplayed enhanced migration of tubules that were connecting andbranching, as well as increased proliferation. Those effects werereduced when rPAI-1_(Δ23) and scuPA were added separately, butsimultaneously to the cultured cells. However, rPAI-1₂₃ was able tomaintain its inhibitory effect on tubule formation in MATRIGEL™.Following rPAI-l₂₃ treatment, single endothelial cells were embedded inthe MATRIGEL™, but there was very little migration and proliferation ofthose cells. When uPA (single chain or two chain) and rPAI-1₂₃ werefirst reacted and then added to the BAEC, there was a small increase intubule formation, extension and proliferation from the focal centers.The tubules that formed were spindly. When rPAI-1₂₃ and uPA were addedseparately but simultaneously, there was less proliferation andextension than observed in the cells that were treated with the reactionmixture. Overall, there was very little change in the anti-angiogeniceffects of rPAI-1₂₃ when uPA was also added exogenously.

[0058] Migration of tubules appeared to be VEGF-dependent. By day 4, therPAI-1₂₃-VEGF-treated rings displayed a reduction in the rate ofmigration similar to that measured in the BBE-treated samples. There wasa significant difference in the structure of the VEGF-treated controltubules and the rPAI-1₂₃-VEGF-treated samples. VEGF-treated rings had adiffuse branching pattern with branches coming off at right angles,whereas the rPAI-1₂₃-VEGF-treated rings formed a very tightly packedmass of tubules that extended in parallel. These data show that rPAI-1₂₃and rPAI-1_(Δ23) are able to inhibit the migratory function of newsprouts from chick aortic rings stimulated with BBE. The rPAI-1₂₃protein inhibits the migration of new sprouts stimulated with VEGF. Inthe rPAI-1₂₃ and rPAI-1_(Δ23)-treated aortic rings apoptosis ofsprouting endothelial cells was observed. The apoptosis appeared toresult in breakage of the tubule. Hence, in a further embodiment of thepresent invention, rPAI-1 proteins lacking a reactive center loop andlacking at least a portion of a heparin-binding domain may be used in amethod for inhibiting cell migration. In a preferred embodiment, arPAI-1 protein for use in inhibiting cell migration is rPAI-1₂₃ orrPAI-1_(Δ23).

[0059] The modulation of VEGF from a complex with heparin by rPAI-1proteins was analyzed. Variable concentrations of each rPAI-1 proteinwere incubated with a VEGF-heparin complex to examine the release and/oractivation of VEGF-A. VEGF activation and release from the complexeswere then tested in the presence of activator molecules uPA or plasmin.Western blot analysis of the complexes was conducted with an antibodyagainst heparin-binding VEGF-A isoforms.

[0060] Experiments were conducted with the VEGF-A_(165,189,206) isoformin an rPAI-1-VEGF-heparin complex containing activator molecules uPA orplasmin. When rPAI-1₂₃ was part of the complex, there were only tracesof the 46 kDa activated VEGF-A_(165,189,206). In the presence ofrPAI-1_(Δ23), VEGF was released from heparin in the absence or presenceof either uPA or plasmin. Similarly, the presence of rPAI-1₂₄ in themixture with VEGF-heparin resulted in the release of active VEGF at allconcentrations of rPAI-1₂₄. In the rPAI-1₂₄-containing reactions, theVEGF release occurred in the presence of uPA or plasmin. Reactionmixtures containing rPAI-1_(Hep23) showed activated VEGF in reactionscontaining low concentrations of rPAI-1_(Hep23) in the absence orpresence of either uPA or plasmin. At higher concentrations ofrPAI-1_(Hep23), there was a blockage of VEGF release and an increase inVEGF-containing fragments between 60 and 80 kDa. The high molecular massfragments containing VEGF had cross-reactivity with VEGF-B₁₈₆ and wereindicative of the 60-62 kDa active VEGF-B₁₈₆ homodimer (Cao, et al.(1999) supra). The greater than 80 kDa molecular mass containing VEGFalso existed in samples containing rPAI-1₂₃, rPAI-1_(Δ23), and rPAI-124.However, the samples containing rPAI-1_(Hep23) clearly had a greateramount of VEGF-A complexed at a high molecular mass. These experimentsshowed that rPAI-1_(Δ23) and rPAI-1₂₄ do not block the activation orrelease of VEGF from a complex with heparin. Although rPAI-1_(Δ23) andrPAI-1₂₄ both have a partial heparin-binding domain, only rPAI-1₂₄ hasRCL. The RCL may alter the conformation such that the partial heparindomain in rPAI-1₂₄ is obscured and unable to block the release ofactivated VEGF. On the other hand, both rPAI-1₂₃ and rPAI-1_(Hep23) areable to block the release of VEGF from a complex with heparin. Thesedata indicate that the heparin-binding domain in each of these twoisoforms participates in blocking the release of active VEGF-A and thatpartial heparin-binding is more effective in blocking VEGF-A activationand/or release. These findings indicate that the ability of rPAI-1₂₃ toblock growth and vessel sprouting may be due to its ability to blockVEGF activation.

[0061] Experiments were also conducted to determine which VEGF isoformswere complexed with heparin-rPAI-1₂₃ at the high molecular mass as seenin the previous experiments. For these experiments, DTT was added to thefinal reaction mixture containing VEGF-heparin-rPAI-1₂₃+uPA (or plasmin)for 2.5 hour at 37° C. The rPAI-1₂₃-VEGF-heparin complexes containedmultiple-sized VEGF fragments representative of mature or activeVEGF-A_(165,189): the fragment less than 34 kDa observed in thisexperiment corresponded to activated VEGF-A₁₈₉ (Plouet, et al. (1997)supra; Keyt, et al. (1996) J. Biol. Chem. 271:7788-7795); the 34-38 kDafragments corresponded to plasmin or uPA activated VEGF-A_(165,189)Plouet, et al. (1997) supra; Keyt, et al. (1996) J. Biol. Chem.271:7788-7795); the 40-45 kDa fragments corresponded to uPA-maturedVEGF-A₁₈₉ or VEGF-A₁₆₅ (Plouet, et al. (1997) supra; Keyt, et al. (1996)J. Biol. Chem. 271:7788-7795) and the 50-52 kDa band was the molecularmass of mature, but not active VEGF (Plouet, et al. (1997) supra).

[0062] In VEGF-heparin complexes containing lower concentrations ofrPAI-1₂₃ protein, there were activated VEGF fragments between 30 and 42kDa which corresponded to the reported sizes for processedVEGF-A_(165,189,206). The fragments that migrated at 36-38 kDa were alsoin reactions containing uPA or plasmin. The most predominant VEGFfragment released from a VEGF-heparin-rPAI-1₂₃ complex was seen near 50kDa (mature VEGF) in the absence of uPA and plasmin. When the rPAI-1₂₃protein concentration was increased to 30 nM, the active VEGF at 50 kDawas absent and the products were approximately 30, 38-42 kDa whichcorresponded in size to uPA-matured VEGF-A₁₈₉ and VEGF-A₁₆₅, orplasmin-activated VEGF-A₁₈₉. In mixtures containing aVEGF-heparin-rPAI-1_(Hep23) complex, the associated VEGF remained at ahigher molecular mass (greater than 80 kDa), except when rPAI-1_(Hep23)was at a low concentration. The rPAI-1_(Hep23) protein conformationmaintained VEGF-A_(165,189,206) in a complex with heparin. Therefore,the rPAI-1₂₃ may be used to block the release of multiple forms ofmatured, activated, and processed (uPA and plasmin cleaved) VEGF-Afragments reported for heparin-binding VEGF-A_(165,189) isoforms.

[0063] Analysis of VEGF-A contained within the culture medium ofrPAI-1-treated endothelial cells was also conducted. TherPAI-1₂₃-treated cells primarily contain VEGF-A fragments at a molecularmass greater than 50 kDa. A small fraction of VEGF fragments at amolecular mass less than 50 kDa were also observed in therPAI-1₂₃-treated samples. The fragments greater than 50 kDa wererepresentative of mature or pro-VEGF and those less than 50 kDacorresponded to active VEGF. In the culture medium samples collectedrPAI-1_(Δ23)-treated cells there was an abundance of VEGF-A fragments ata molecular mass of ˜36-45 kDa at all time points (6, 15, 24, 30, 48,and 72 hours). This molecular mass corresponded to active or uPA/plasminprocessed, active VEGF-A. All rPAI-1_(Δ23)-treated samples alsocontained mature or pro-VEGF at a molecular mass greater than 50 kDa.One of the inactive fragments in the 15 hour time point sample wasabsent in samples at 6, 24, 30, 48, and 72 hours. The culture media fromall untreated and rPAI-1₂₄-treated cells, contained active VEGF and asmall amount of processed, activate VEGF. The culture medium samplesfrom the rPAI-1_(Hep23)-treated cells primarily contained inactive VEGFand lesser amounts of processed, active VEGF. The results of theseexperiments clearly showed that VEGF in rPAI-1_(Δ23)-treated culturemedium contains a much greater amount of active VEGF, mostlyrepresentative of uPA or plasmin processed VEGF. Similar VEGF fragmentswere absent or present to a lesser degree in the untreated or rPAI-1₂₃—,rPAI-1_(Hep23)-, and rPAI-1₂₄-treated cells. The culture media fromrPAI-1₂₄-treated and untreated cells contained a greater amount ofactive, unprocessed VEGF than the media from rPAI-1₂₃, rPAI-1_(Δ23)-, orrPAI-1_(Hep23)-treated cells. These data corresponded with thebiochemical analysis of VEGF released from a complex with heparin andrPAI-1 proteins. The molecular mass of all of the VEGF fragmentscorresponded to dimeric VEGF-A despite the rigorous reducing conditionsapplied to all samples.

[0064] Differences in heparan sulfate-bound VEGF-A isoforms inrPAI-1-treated cells were analyzed using two different antibodies.Immunoblots were probed for VEGF-A with an antibody specific forepitopes common to VEGF-A_(165,189,205). The results of theseexperiments showed that, at the 12-hour time point, the rPAI-1_(Hep23)-and rPAI-1₂₄-treated cells contained two fragments of VEGF-A at amolecular mass near 58 kDa. In the rPAI-1₂₃-treated,rPAI-1_(Δ23)-treated, and untreated samples, VEGF-A was absent or barelyvisible. However, when two competing antibodies specific for VEGF-A weresimultaneously incubated with immunoblots containing proteins releasedfrom a heparinase digest, the rPAI-1₂₃-treated, rPAI-1_(Δ23)-treated,and untreated cells show VEGF-A released from heparan sulfate. Among thesamples, the rPAI-1₂₃-treated cells at the six-hour time point had thegreatest amount of detectable VEGF-A released in the enzymatic digest.The VEGF was seen as two distinct fragments with a small difference inmolecular mass. Each fragment corresponded to the molecular mass ofdimeric VEGF, despite the rigorous reducing conditions. By 12 hours,VEGF was not detected in rPAI-1₂₃-treated cells. However, at the 12-hourtime point, the rPAI-1_(Δ23)-treated and the untreated cells showed avariable molecular mass in VEGF released as a result of the digest. TheVEGF fragments in the rPAI-1₂₃-treated samples were very close to 50kDa, whereas, the VEGF fragments in the untreated and therPAI-1_(Δ23)-treated cells corresponded to a molecular mass that wasmore representative of VEGF-A₁₈₉ or VEGF-A₂₀₆. The rPAI-1_(Hep23)- andrPAI-1₂₄-treated cells did not show the release of heparan sulfate-boundVEGF when the blot was probed with competing antibodies. The results ofthese sets of experiments showed that different VEGF-A isoforms werereleased with the heparinase digest, depending upon the rPAI-1treatment. The use of competing antibodies exposed binding sites inrPAI-1₂₃-treated, rPAI-1_(Δ23)-treated, and untreated cells that werenot detected with the single antibody probe for VEGF-A_(165,189,205).The most pronounced difference was the intensity and molecular mass ofVEGF-A at the six-hour time point in rPAI-1₂₃-treated cells. Itcorresponded to a molecular mass near that reported for dimericmature/pro-VEGF-A₁₆₅ and/or mature VEGF-A₁₈₉. Because the VEGF fragmentsbecame intensely visible upon competition with an antibody specific forthe active site of VEGF-A₁₆₅, the data indicated that the fragmentsslightly above 50 kDa were VEGF-A₁₆₅. These data also showed that theconformation of VEGF was altered as a result of rPAI-1₂₃ treatment.Also, the presence of extracellular matrix-associated VEGF-A at the6-hour time point was coordinate with the absence of activated VEGF inthe culture medium of rPAI-1₂₃-treated cells at 6 hours. The VEGF-Afragments that become visible at the 12 hour time points in untreated orrPAI-1_(Δ23)-treated cells were at a greater molecular masscorresponding to pro-VEGF-A₁₈₉ or pro-VEGF-A₂₀₆, the isoforms that hadthe greatest affinity for heparan sulfate. Similarly, the VEGF detectedin all samples was representative of dimeric VEGF, despite the reducingconditions.

[0065] Additional studies were conducted to more clearly define themechanism by which rPAI-1₂₃ prevents the release of VEGF from heparansulfate. Taking into consideration that rPAI-1₂₃, uPA and VEGF havebinding domains for heparin/heparan sulfate; rPAI-1₂₃ binds uPA at theadditional uPA binding domain; and uPA can bind to and activatepro-VEGF; it was determined whether rPAI-1₂₃ was retaining VEGF in acomplex with uPA. Primarily, it was determined whether treatment withrPAI-1₂₃ could alter the binding affinity of VEGF for a specific heparansulfate chain. Moreover, it was determined whether an rPAI-1₂₃ oruPA-rPAI-1₂₃ complex prevented the release of VEGF from a specificheparan sulfate chain. Thus, BAEC were treated either withanti-angiogenic rPAI-1₂₃ protein, rPAI-1₂₃ complexed with uPA, orvarious pro-angiogenic molecules (VEGF, uPA, bFGF, rPAI-1_(Hep23)) for16 hours at 37° C. The treatment molecules were removed using Hanks'balanced salt solution (HBSS) washes and subsequently 1 Unit/mL ofeither heparinase I or heparinase III was added to the treated cells for1 hour. Proteins released by heparinase treatment were collected andequivalent amounts of protein were separated in a 4-20% SDS reducingpolyacrylamide gel. Immunoblots containing the separated proteins wereprobed with two competing VEGF-specific antibodies. In cells treatedwith rPAI-1₂₃, activated VEGF (monomer at 16 kDa) was not released fromheparan sulfate during heparinase I treatment, rather inactive and/orcomplexed VEGF was observed near 50 and 80 kDa. Similarly, VEGF was notreleased from heparan sulfate when cells were treated with rPAI-1₂₃/uPAcomplex. However, if uPA and rPAI-1₂₃ were added separately, butsimultaneously, to the culture medium of endothelial cells, VEGF wasreleased from heparan sulfate by heparinase I treatment. Heparinase IIItreatment also did not liberate VEGF from cells treated with anrPAI-1₂₃/uPA complex, however, VEGF was released from cells treated withrPAI-1₂₃. Thus, there is more VEGF associated with heparan sulfatecontaining side chains which may be liberated by heparinase I digestionas compared to heparinase III digestion. In addition, cells treated withpro-angiogenic rPAI-1_(Hep23) have a much greater amount of releasedactive VEGF at a molecular mass between 34 and 50 kDa than any of theother pro-angiogenic molecules (uPA, VEGF, or bFGF). However, treatingcells with a rPAI-1_(Hep23)/uPA complex blocks the release active VEGFfrom heparan sulfate. Accordingly, these data indicate that anrPAI-1₂₃/uPA complex is involved in the inhibition of VEGF release fromheparan sulfate and uPA can inhibit the release of VEGF when complexedwith rPAI-1_(Hep23).

[0066] Subsequent analysis was conducted to examine VEGF binding tosites on heparan sulfate and to VEGF receptors. Endothelial cells werefirst treated with 0.6 nM rPAI-1₂₃ or 10 ng/mL VEGF for 2, 4 and 16hours. Subsequently, the cells were placed on ice and washed withice-cold phosphate buffered saline (PBS) before the addition of¹²⁵I-VEGF. After a one-hour incubation with ¹²⁵I-VEGF on ice, cells werewashed extensively with ice-cold PBS containing 0.05% bovine serumalbumin. The cells were then washed with 0.35 M NaCl for 10 minutes toremove VEGF bound to heparan sulfate. The cells were also washed withice-cold PBS containing 0.05% BSA before the addition of 1 N NaOH for 30minutes. Fractions containing released VEGF or dissolved membraneproteins (i.e., dissolved in 1 N NaOH) were collected and prepared forcounting the iodinated VEGF. Further, triplicate samples of each testand time point were prepared to perform a cell count. The cell count wasused to normalize the DPM with cell number.

[0067] The results of these experiments showed that following 16 hoursof treatment, the number of available VEGF binding sites on heparansulfate in rPAI-1₂₃-treated endothelial cells were more than three timesthat of the untreated endothelial cell control. In contrast, the numberof receptor binding sites on rPAI-1₂₃-treated cells was very similar tothat of the untreated cells, indicated that VEGF was bound to availablereceptors. Competition assays with cold VEGF verified that the labelingwas VEGF-specific.

[0068] In vitro experiments were conducted with Annexin V to measureapoptosis in bovine and porcine aortic endothelial cells treated withrPAI-1₂₃ protein. These data showed that rPAI-1₂₃ treatment resulted in300% more apoptotic bovine aortic endothelial cells (BAEC) as comparedto porcine aortic endothelial cells (PAEC). Since PAEC do not expressVEGFR-1 or VEGFR-2, these experiments clearly showed that most, but notall, of the apoptosis induced by rPAI-1₂₃ was through a mechanism thatblocked/inhibited VEGF receptors 1 and/or 2.60% of the apoptosis inrPAI-1₂₃-treated BAEC could be attributed to inhibition of VEGF-VEGFRfunctional activity. These data demonstrate a mechanism whereby rPAI-123blocked release of active VEGF-A thereby preventing (or limiting)binding to VEGF receptors to result in apoptosis. The 65% reduction inmigration of VEGF-stimulated tubules from rPAI-1₂₃-treated embryonicchick aortic rings provides a mechanism which inhibits VEGFR-2 function.Accordingly, in a further embodiment of the present invention, rPAI-1proteins used to block the release of VEGF from a VEGF-heparin complexlack an RCL domain and lack all or at least a heparin-binding domain asfound in, for example, rPAI-1₂₃, rPAI-1₂₄ and rPAI-1_(Hep23). In apreferred embodiment, rPAI-1₂₃ is used to block the release of VEGF froma VEGF-heparin complex.

[0069] Further, in vitro experiments were conducted to evaluate theeffect of rPAI-1₂₃ on MT1-MMP-mediated cellular phenotypes. Theup-regulated expression of VEGF by MT1-MMP is associated with aninvasive phenotype, particularly on a type I collagen matrix (Murakami,et al. (1999) Neoplasia 1(5):424-30; Seiki (2003) Cancer Lett.194(1):1-11). Thus, the activity of rPAI-1₂₃ was evaluated usingendothelial cells plated onto a type I collagen matrix. Endothelialcells were seeded onto 6-well culture plates and allowed to adhere for 1hour before the addition of a type I collagen coat over the cells. Theplates were incubated at 37° C. for 1 hour to allow the collagen tocongeal. Dulbecco's Modified Eagle's Medium containing VEGF (10 ng/mL),rPAI-1₂₃ (0.6 nM) or rPAI-1_(Hep23) (0.6 nM) was added to each well andcultures were incubated at 37° C. for seven days; medium containing theanti- or pro-angiogenic molecules was replaced every 48 hours.

[0070] Most BAECs treated with rPAI-1₂₃ were apoptotic within the first24 hours and looked the same throughout the seven day incubation.Conversely, rPAI-1_(Hep23)- and VEGF-stimulated BAECs formed tubes onthe collagen matrix and the untreated cells formed incomplete tubes.These data are consistent with the RNase protection assays which showedthat anti-angiogenic rPAI-1₂₃ down-regulated MT1-MMP and pro-angiogenicrPAI-1_(Hep23) and VEGF up-regulated MT1-MMP. The extensive apoptosisinduced by rPAI-1₂₃ by 24 hours indicates a possible mechanism by whichrPAI-1₂₃ reduces the invasiveness of tumors. The in vivo physiologicalrelevance of the various cleaved isoforms of PAI-1 was examined inpatient samples isolated from two cardiovascular clinical studies. Inone cardiology study, patients were evaluated for diseased vessels andthe number of collaterals. Blood was drawn from said patients and plasmawas examined for cleaved PAI-1. Thirty-five patient samples wereexamined. Of these patients, some had cleaved PAI-1 in the plasma at amolecular mass near 28 kDa and some had no observable cleaved PAI-1. Allsamples examined had intact PAI-1 of variable intensities. In somesamples there was also evidence of a cleavage product at a molecularmass close to intact PAI-1. This fragment size has been reported in invitro studies and is correlated with thrombin.

[0071] Plasminogen kringle domains 1-3 were also examined in thesepatient samples. A correlation between the presence of cleaved PAI-1near 28 kDa and plasminogen kringles 1-3 at 34 kDa was observed.

[0072] In a second clinical study, atherosclerotic plaques weresurgically-removed. Proteins were isolated from the plaques by tissuehomogenization on ice in the presence of protease inhibitors or fromproteins released from plaque cultures. Subsequently, the proteins wereanalyzed for cleaved PAI-1 and plasminogen cleavage products. CleavedPAI-1 near 28 kDa was present in all examined plaques at variableamounts. Further, there was an enormous difference in plasminogencleavage products detected with kringle 1-3 antibody probe. In somesamples, fragments near 50 kDa were observed. In other samples, doubletsbetween 28 and 34 kDa were observed. Moreover, cleaved plasminogen wasabsent or barely detectable in some samples.

[0073] PAI-1 can be cleaved at the P1 and P′1 residues of the RCL,resulting in a substrate with a molecular mass of 39 kDa (Dhanabal, etal. (1999) J. Biol. Chem. 274:11721-11726; Aleshkov, et al. (1996) J.Biol. Chem. 271:21231-21238; Declerck, et al. (1992) J. Biol. Chem.267:11693-11696). The amount of substrate PAI-1 (RCL cleaved, inactive)is increased during the interaction with PAI-1 with thrombin in thepresence of heparin and vitronectin (Van Meijer, et al. (1997) Blood90:1874-1882). A smaller, cleaved PAI-1 fragment (<31 kDa) has beenshown to be produced as a result of addition of heparin and thrombin toa pre-existing PAI-1/thrombin complex (Patston and Schapira (1994) Blood84:1164-1172). The deletions at the carboxy-terminus exclude the RCL inthree of the four rPAI-1 isoforms described herein and each of theseproteins function differently with respect to pro- and anti-angiogenicmechanisms. Therefore, the functional activity of these proteins is notsolely dependent upon the absence of the reactive center loop. Thestructural difference in rPAI-1₂₃, rPAI-1_(Δ23), and rPAI-1_(Hep23) isthe heparin (heparan sulfate)-binding domain which accounts for thedifferences in functional activity of the three proteins. The novelfunctional activity of these isoforms of PAI-1 makes them useful indifferentially modulating angiogenesis. In a preferred embodiment of thepresent invention, an rPAI-1 isoform lacking an RCL domain and lacking aleast a portion of the heparin-binding domain is useful in a method ofblocking, reducing or decreasing angiogenesis. Exemplary isoformslacking an RCL domain and at least a portion of the heparin-bindingdomain include, but are not limited to, rPAI-1₂₃ and rPAI-1_(Δ23). Inanother preferred embodiment of the present invention, an rPAI-1 isoformlacking an RCL domain and containing a complete heparin-binding domainis useful in a method of stimulating or increasing angiogenesis. Anexemplary isoform lacking an RCL domain and containing a completeheparin-binding domain includes, but is not limited to, rPAI-1_(Hep23).

[0074] The rPAI-1₂₃, rPAI-1_(Hep23) and rPAI-1_(Δ23) isoforms of thepresent invention are useful in treating diseases or processes that aremediated by, or involve, angiogenesis. The present invention provides amethod of treating an angiogenesis-mediated disease with an effectiveamount of a PAI-1 isoform lacking the RCL domain and either containing acomplete heparin-binding domain or lacking at least a portion of theheparin-binding domain. It is contemplated that rPAI-1 isoforms lackingthe RCL domain and lacking at least a portion of the heparin-bindingdomain, e.g., rPAI-1123 and rPAI-1₂₃, would be useful as anti-angiogenicagents. Angiogenesis-mediated diseases for which anti-angiogenic agentswould be useful in alleviating the signs or symptoms of include, but arenot limited to, solid tumors; blood bourne tumors such as leukemias;tumor metastasis; benign tumors, for example hemangiomas, acousticneuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoidarthritis; psoriasis; ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation. Anti-angiogenic agents are also useful in thetreatment of diseases that have angiogenesis as a pathologic consequencesuch as cat scratch disease (Rochele minalia quintosa) and ulcers(Helobacter pylori). Furthermore, PAI-1 isoforms lacking the RCL domainand lacking at least a portion of the heparin-binding domain areeffective in the treatment of disease of excessive or abnormalstimulation of endothelial cells. These diseases include, but are notlimited to, intestinal adhesions, atherosclerosis, scleroderma, andhypertrophic scars, i.e., keloids.

[0075] Further contemplated is rPAI-1 isoforms lacking the RCL domainand containing a complete heparin-binding domain, e.g., rPAI-1_(Hep23),as proangiogenic agents. Proangiogenic agents would be useful forstimulating wound healing, replacing clogged arteries to improvecirculation in patients with arterial clogging, and treating varioustypes of heart disease to promote the growth of blood vessels therebyreducing the need for bypass surgery.

[0076] While specific PAI-1 isoforms are disclosed herein, it would bewell within the ability of the skilled artisan, using the resultsprovided by this disclosure, to generate additional PAI-1 isoformslacking at least a portion of the heparin-binding domain. For example,one could remove residue Lys-65 and/or Lys-69 of the rPAI-1_(Hep23)isoform to decrease the proangiogenic activity of this isoform.

[0077] In general, those skilled in the art will appreciate that minordeletions or substitutions may be made to the amino acid sequences ofrPAI-1 isoforms of the present invention without unduly adverselyaffecting the activity thereof. Thus, isoforms containing such deletionsor substitutions are also contemplated. In isoforms containingsubstitutions or replacements of amino acids, one or more amino acids ofa peptide sequence may be replaced by one or more other amino acidswherein such replacement does not affect the function of that sequence.Such changes can be guided by known similarities between amino acids inphysical features such as charge density, hydrophobicity/hydrophilicity,size and configuration, so that amino acids are substituted with otheramino acids having essentially the same functional properties. Forexample: Ala may be replaced with Val or Ser; Val may be replaced withAla, Leu, Met, or Ile, preferably Ala or Leu; Leu may be replaced withAla, Val or Ile, preferably Val or Ile; Gly may be replaced with Pro orCys, preferably Pro; Pro may be replaced with Gly, Cys, Ser, or Met,preferably Gly, Cys, or Ser; Cys may be replaced with Gly, Pro, Ser, orMet, preferably Pro or Met; Met may be replaced with Pro or Cys,preferably Cys; His may be replaced with Phe or Gln, preferably Phe; Phemay be replaced with His, Tyr, or Trp, preferably His or Tyr; Tyr may bereplaced with His, Phe or Trp, preferably Phe or Trp; Trp may bereplaced with Phe or Tyr, preferably Tyr; Asn may be replaced with Glnor Ser, preferably Gln; KGln may be replaced with His, Lys, Glu, Asn, orSer, preferably Asn or Ser; Ser may be replaced with Gln, Thr, Pro, Cysor Ala; Thr may be replaced with Gln or Ser, preferably Ser; Lys may bereplaced with Gln or Arg; Arg may be replaced with Lys, Asp or Glu,preferably Lys or Asp; Asp may be replaced with Lys, Arg, or Glu,preferably Arg or Glu; and Glu may be replaced with Arg or Asp,preferably Asp. Once made, changes can be routinely screened todetermine their effects on function.

[0078] The rPAI-1 isoforms of the present invention may be purified fromhost cells which express the same, in accordance with known techniques,or even manufactured synthetically. Alternatively, the rPAI-1 isoformsmay be used as part of a gene therapy approach.

[0079] Recombinant PAI-1 proteins may be produced using methodsexemplified herein for using other well-known methods for long-term,high-yield production of recombinant proteins. For example, nucleic acidsequences encoding, e.g., human, porcine, or bovine PAI-1, may berecombinantly engineered, using well-known methods, to produce therPAI-1 isoforms of the present invention which lack the RCL domain andcontain a complete heparin-binding domain or lack at least a portion ofa heparin-binding domain as defined herein. A recombinant nucleic acidsequence encoding a rPAI-1 isoform may then be incorporated into anexpression vector. An expression vector is a replicable DNA construct inwhich a nucleic acid sequence encoding an rPAI-1 isoform of the presentinvention is operably linked to suitable control sequences capable ofeffecting the expression of the isoform in a suitable host. The need forsuch control sequences will vary depending upon the host selected andthe transformation method chosen. Generally, control sequences include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation.

[0080] Vectors comprise plasmids, viruses (e.g., adenovirus,cytomegalovirus), phage, retroviruses and integratable DNA fragments(i.e., fragments integratable into the host genome by recombination).The vector replicates and functions independently of the host genome, ormay, in some instances, integrate into the genome itself. Expressionvectors should contain a promoter and RNA binding sites that areoperably linked to the gene to be expressed and are operable in the hostorganism.

[0081] Suitable host cells include prokaryotes, yeast cells, or highereukaryotic organism cells. Prokaryote host cells include gram negativeor gram positive organisms, for example Escherichia coli (E. coli) orBacilli. Higher eukaryotic cells include established cell lines ofmammalian origin. Exemplary host cells are E. coli W3110 (ATCC 27,325),E. coli B, E. coli X1776 (ATCC 31,537), E. coli 294 (ATCC 31,446). Abroad variety of suitable prokaryotic and microbial vectors areavailable. E. coli is typically transformed using pBR322. See Bolivar,et al., ((1977) Gene 2:95). Promoters most commonly used in recombinantmicrobial expression vectors include the beta-lactamase (penicillinase)and lactose promoter systems (Chang, et al. (1978) Nature 275:615;Goeddel, et al. (1979) Nature 281:544), a tryptophan (trp) promotersystem (Goeddel, et al. (1980) Nucl. Acids Res. 8:4057; EP36,776) andthe tac promoter (De Boer, et al. (1983) Proc. Natl. Acad. Sci. USA80:21). The promoter and Shine-Dalgarno sequence (for prokaryotic hostexpression) are operably linked to the DNA of the present invention,i.e., they are positioned so as to promote transcription of themessenger RNA from the DNA.

[0082] Eukaryotic microbes such as yeast cultures may be transformedwith suitable expression vectors as exemplified herein. Alternatively,Saccharomyces cerevisiae may be used. Yeast vectors may contain anorigin of replication from the 2 micron yeast plasmid or an autonomouslyreplicating sequence (ARS), a promoter, DNA encoding the desiredprotein, sequences for polyadenylation and transcription termination,and a selection gene. An exemplary plasmid is YRp7, (Stinchcomb, et al.(1979) Nature 282:39; Kingsman, et al. (1979) Gene 7:141; Tschemper, etal. (1980) Gene 10:157). This plasmid contains the trp1 gene, whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones (1977) Genetics 85:12). The presence of the trp1 lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.

[0083] Cultures of cells derived from multicellular organisms are alsodesirable hosts for recombinant protein synthesis. In principal, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture, including insect cells. Propagation of such cellsin cell culture has become a routine procedure. See Tissue Culture,Academic Press, Kruse and Patterson, editors (1973). Examples of usefulhost cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO)cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines. Expressionvectors for such cells ordinarily include (if necessary) an origin ofreplication, a promoter located upstream from the gene to be expressed,along with a ribosome binding site, RNA splice site (ifintron-containing genomic DNA is used), a polyadenylation site, and atranscriptional termination sequence.

[0084] The transcriptional and translational control sequences inexpression vectors to be used in transforming vertebrate cells are oftenprovided by viral sources. For example, commonly used promoters arederived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40). See,e.g., U.S. Pat. No. 4,599,308. The early and late promoters are usefulbecause both are obtained easily from the virus as a fragment that alsocontains the SV40 viral origin of replication. See Fiers, et al. (1978)Nature 273:113. Further, the protein promoter, control and/or signalsequences may also be used, provided such control sequences arecompatible with the host cell chosen.

[0085] An origin of replication may be provided either by constructionof the vector to include an exogenous origin, such as may be derivedfrom SV40 or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV),or may be provided by the host cell chromosomal replication mechanism.If the vector is integrated into the host cell chromosome, the lattermay be sufficient.

[0086] Host cells such as insect cells (e.g., cultured Spodopterafrugiperda cells) and expression vectors such as the baculorivusexpression vector (e.g., vectors derived from Autographa californicaMNPV, Trichoplusia ni MNPV, Rachiplusia ou MNPV, or Galleria ou MNPV)may be employed to make proteins useful in carrying out the presentinvention, as described in U.S. Pat. Nos. 4,745,051 and 4,879,236. Ingeneral, a baculovirus expression vector comprises a baculovirus genomecontaining the gene to be expressed inserted into the polyhedrin gene ata position ranging from the polyhedrin transcriptional start signal tothe ATG start site and under the transcriptional control of abaculovirus polyhedrin promoter.

[0087] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding an rPAI-1 may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing the rPAI-1 in infected host cells (Loganand Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0088] Rather than using vectors that contain viral origins ofreplication, one may transform mammalian cells by the method ofcotransformation with a selectable marker and the chimeric protein DNA.An example of a suitable selectable marker is dihydrofolate reductase(DHFR) or thymidine kinase. See U.S. Pat. No. 4,399,216. Such markersare proteins, generally enzymes, that enable the identification oftransformant cells, i.e., cells which are competent to take up exogenousDNA. Generally, identification is by survival or transformants inculture medium that is toxic, or from which the cells cannot obtaincritical nutrition without having taken up the marker protein.

[0089] Depending on the host cell to be transformed, any well-knownmeans of introducing the expression vector containing nucleic acidsequences encoding an rPAI-1 may be used. Following the introduction ofthe expression vector, cells may be allowed to grow for 1-2 days in anenriched media before they are switched to selective media. The purposeof the selectable marker is to confer resistance to selection, and itspresence allows growth and recovery of cells that successfully expressthe introduced sequences. Resistant clones of stably transformed cellsmay be proliferated using tissue culture techniques appropriate to thecell type.

[0090] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, et al. (1977) Cell11:223-32) and adenine phospho-ribosyltransferase (Lowy, et al. (1980)Cell 22:817-23) genes which can be employed in tk− or aprt− cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman and Mulligan (1988)Proc. Natl. Acad. Sci. 85:8047-51). Alternatively, visible such asanthocyanins, β glucuronidase and its substrate GUS, and luciferase andits substrate luciferin, may be used not only to identify transformants,but also to quantify the amount of transient or stable proteinexpression attributable to a specific vector system (Rhodes, et al.(1995) Methods Mol. Biol. 55:121-131).

[0091] Host cells transformed with nucleotide sequences encoding anrPAI-1 may be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode an rPAI-1 may be designed to contain signal sequences whichdirect secretion of the rPAI-1 through a prokaryotic or eukaryotic cellmembrane. Other constructions may be used to join sequences encoding anrPAI-1 to nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and the rPAI-1 may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing an rPAI-1 and a nucleic acid encoding sixhistidine residues preceding a thioredoxin or an enterokinase cleavagesite. The histidine residues facilitate purification on IMAC(immobilized metal ion affinity chromatography) (see, e.g., Porath, etal. (1992) Prot. Exp. Purif. 3:263-281) while the enterokinase cleavagesite provides a means for purifying the rPAI-1 from the fusion protein.A discussion of vectors which contain fusion proteins is provided inKroll, et al. (1993) DNA Cell Biol. 12:441-453).

[0092] In addition to recombinant production of the rPAI-1 isoforms maybe produced by direct peptide synthesis using solid-phase techniques(Merrifield (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of the rPAI-1isoforms may be chemically-synthesized separately and combined usingchemical methods to produce the full-length molecule.

[0093] Recombinant proteins provided herein may be used as isolated andsubstantially purified proteins in pharmaceutically acceptableformulations using formulation methods known to those of ordinary skillin the art. These formulations can be administered by standard routes.In general, the combinations may be administered by the topical,transdermal, intraperitoneal, intracranial, intracerebroventricular,intracerebral, intravaginal, intrauterine, oral, rectal or parenteral(e.g., intravenous, intraspinal, subcutaneous or intramuscular) route.In addition; the PAI-1 proteins may be incorporated into biodegradablepolymers allowing for sustained release of the proteins, the polymersbeing implanted in the vicinity of where drug delivery is desired, forexample, at the site of a tumor or implanted so that the rPAI-1 isoformis slowly released systemically. Osmotic minipumps may also be used toprovide controlled delivery of high concentrations of rPAI-1 throughcannulae to the site of interest, such as directly into a metastaticgrowth or into the vascular supply to that tumor. The biodegradablepolymers and their use are described, for example, in detail in Brem etal. ((1991) J. Neurosurg. 74:441-446).

[0094] It is contemplated that the rPAI-1 isoforms of the presentinvention may be co-administered. For example, rPAI-1₂₃ and rPAI-1_(Δ23)may be used in a combination therapy for enhancing the formation ofangiostatin and/or modulating angiogenesis.

[0095] The rPAI-1 formulations may conveniently be presented in unitdosage form and may be prepared by conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the active ingredient and the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0096] Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for example,water for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

[0097] It is further contemplated that the rPAI-1 isoforms may beadministered via stem cells which are genetically engineered to producethe rPAI-1 isoform.

[0098] Cells to be targeted by the rPAI-1 protein formulations include,but are not limited to, an endothelial cell, a lymphocyte, a macrophage,a glia cell, a fibroblast, a liver cell, a kidney cell, a muscle cell, acell of the bone or cartilage tissue, a synovial cell, a peritonealcell, a skin cell, an epithelial cell, a leukemia cell or a tumor cell.

[0099] The present invention provides administration of an “effectiveamount” of rPAI-1 proteins to modulate angiogenesis or angiogenicprocesses or treat angiogenesis-mediated diseases. An “effective amount”is considered an amount of protein empirically determined to benecessary to achieve a reproducible change in cell proliferation orapoptosis (as determined by microscopic or macroscopic visualization andestimation of cell doubling time, or of nucleic acid synthesis assays)or migration (as determined by microscopic or macroscopic visualization)or in reducing the signs or symptoms of an angiogenesis-mediated diseaseas would be understood by one of ordinary skill in the art.

[0100] The specific amount of rPAI-1 protein required by each individualwill be dependent upon the age, health, and weight of the recipient,kind of concurrent treatment, if any, frequency of treatment, and thenature of the effect desired.

[0101] The rPAI-1 isoforms of the present invention may be used topromote in vitro production of blood vessels for transplantation.

[0102] The rPAI-1 isoforms of the present invention are also useful inthe formation of angiostatin containing K1-3 and/or K1-4. Angiostatinformation may be in vivo for therapeutic purposes or in vitro forrecombinant, large-scale production of angiostatin with desiredactivity. It is contemplated that rPAI-1₂₃ and rPAI-1_(Δ23) may be usedalone or in combination to form the desired angiostatin.

[0103] The examples, which follow, are set forth to illustrate methodsof the present invention, and are not to be construed as limitingthereof.

EXAMPLE 1 PAI-1 Gene Deletions

[0104] The DNA encoding the truncated PAI-1 proteins was obtained bydeleting the porcine PAI-1 gene (poPAI-1)(Bijnens, et al. (1997) Thromb.Haemost. 77:350-356; Accession number Y11347). The selection of genefragments was based on the poPAI-1 sequences that correspond to thehuman PAI-1 gene (huPAI-1) (Bosma, et al. (1988) J. Biol. Chem.263:9129-9141; Accession number J03764) sequence reported to code forfunctional domains in human PAI-1 (Reilly and Hutzelmann (1992) supra).Each DNA fragment was isolated from porcine aortic endothelial cells byreverse transcribing RNA into cDNA. The cDNA was made double-stranded ina polymerase chain reaction (PCR) containing porcine PAI-1-specificprimers. Primers for rPAI-1₂₃ amplification are known, for example,Mulligan-Kehoe, et al. ((2001) supra). Primers used to PCR amplify thecDNA into double-stranded DNA coding for rPAI-1_(Δ23) are 5′ primer:5′-GGAATTCATGGATGAGATCAGCACGG-3′ (SEQ ID NO:3) and 3′ primer:5′-GCTCTAGATTTCCACTGGCTGATG-3′ (SEQ ID NO:4). The resulting productcorresponds to nucleotides 471-999 of poPAI-1 and 265-793 of huPAI-1.Likewise, primers used to PCR amplify the cDNA into double-stranded DNAcoding for rPAI-1_(Hep23) are 5′ primer:5′-GGAATTCATGCAGTTCAAGATTGAGGAGAAGGGC-3′ (SEQ ID NO:5) and 3′ primer:5′-GCTCTAGATTTCCACTGGCTGATG-3′ (SEQ ID NO:6). The resulting productcorresponds to nucleotides 390-999 of poPAI-1 and 184-793 of huPAI-1.Furthermore, primers used to PCR amplify the cDNA into double-strandedDNA coding for rPAI-1₂₄ are 5′ primer: 5′-GGAATTCAAGGAGCTCATGG-3′ (SEQID NO:7) and 3′ primer: 5′-GCTCTAGATCAAGGCTCCATCAC-3′ (SEQ ID NO:8). Theresulting product corresponds to nucleotides 444-1346 of poPAI-1 and238-1162 of huPAI-1. Underlined nucleotides denote restriction enzymerecognition sequences. PCR conditions for amplification of all threegenes are known to one skilled in the art, for example, Reilly andHutzelmann ((1992) supra). The PCR-amplified rPAI-1 DNA fragments weredouble-digested with EcoRI and XbaI (Roche, Indianapolis, Ind.) tocreate overhangs. The restricted DNA was ligated into Pichia pastorisyeast shuttle vector pGAPZαA (INVITROGEN™, Carlsbad, Calif.). The TOP 10strain of Escherichia coli was transformed by electroporation asdescribed (Mulligan-Kehoe, et al. (2001) supra). Following an overnightincubation at 37° C., colonies were selected and grown in low salt LBbroth for 5-7 hours at 37° C. The DNA from each colony was isolatedusing a miniprep kit (Qiagen, Inc. Valencia, Calif.) to identify a clonecontaining each gene insert. Positive isolates were identified byrestriction enzyme digests and were verified by sequencing. Eachrecombinant protein was expressed in P. pastoris as described (Reillyand Hutzelmann (1992) supra) and purified by affinity chromatography.The sequences of rPAI-1₂₃, rPAI-1_(Δ23), rPAI-1_(Hep23), and rPAI-1₂₄DNA matched the known sequence of the corresponding segment of porcinePAI-1 DNA. Each rPAI-1 protein corresponded to its expected molecularweight.

EXAMPLE 2 Anti-Angiogenic Effects of rPAI-1 Proteins

[0105] Characterization of the rPAI-1 Protein Interactions with heparin,uPA, and Plasminogen. The functionality of the rPAI-1 proteins was firsttested by incubating each truncated protein with uPA and plasminogen toassess the proteolytic activity of the products of the reaction, asknown in the art, for example Mulligan-Kehoe, et al. ((2001) supra). Thefunctionality of each rPAI-1 isoform was then tested in a reaction withheparin bound to Sepharose beads. First, the rPAI-1₂₃, rPAI-1_(Δ23),rPAI-1_(Hep23), and rPAI-1₂₄ proteins (20 μg) were each incubated with50 μl of heparin-bound Sepharose beads (Amersham Pharmacia Biotech,Piscataway, N.J.) for 2 hours at 37° C. The unbound protein wasseparated from the heparin-Sepharose-bound protein complex bymicrocentrifugation at 4° C. for 15 minutes. The proteins bound to theSepharose beads were washed in TBS/Tween-20 followed by TBS washes.Bound samples were reacted with uPA (0.5 IU/1 μg or rPAI-1 protein) at37° C. for 1 hour followed by a second one hour, 37° C. incubation withplasminogen (1 IU/1 μg of rPAI-1 protein). The reaction samples wereanalyzed on a 15% SDS polyacrylamide gel containing 1.3% casein(zymogram) (Clowes, et al. (1990) Circ. Res. 67:61-67; Allaire, et al.(1998) Circulation 98:249-255). The electrophoresed samples wereincubated overnight at 37° C. in 50 mM Tris-HCl, pH 7.9, buffercontaining 5 mM CaCl₂ to enable plasmin degradation of casein.

[0106] Identification of Plasminogen Kringle Domains. Protein productsfrom the reaction of rPAI-1, uPA and plasminogen were electrophoresed ona 4-20% SDS polyacrylamide gel and transferred to nitrocellulose. Theblots were probed with an antibody (1 μg/ml), specific for plasminogenkringles 1-3 (R&D Systems, Inc., Minneapolis, Minn.). Following a 1hour, room temperature incubation with the primary antibody, a secondaryrabbit anti-goat IgG HC+LC polyclonal antibody (Pierce, Rockford, Ill.)at a concentration of 1 μg/ml was incubated with the anti-kringle-probedmembrane for 1 hour at room temperature. A horseradishperoxidase-conjugated antibody (donkey anti-rabbit IgG, Amersham,Arlington Heights, Ill.) diluted 1:5000 amplified the binding reactionwhich was ultimately detected by addition of a chemiluminescentsubstrate (Amersham Pharmacia).

[0107] In vitro Angiostatin Production in rPAI-1 Treated Cells. Bovineaortic endothelial cells (BAEC) were plated in six-well culture plates.When the cells reached confluence, fresh growth medium containing eitherrPAI-1₂₃ or rPAI-1_(Δ23) (1.2 nM) was added; untreated endothelial cellsserved as the control. The cell culture media and the extracellularmatrix with associated cells were collected from the untreated,rPAI-1₂₃— or rPAI-1_(Δ23)-treated BAEC in a 6, 15, 24, 36, 48, and 72hour time course, using a standard method (Mulligan-Kehoe, et al. (2001)supra). The concentration of protein in each sample from the time coursewas measured in a Bradford assay (Bradford (1976) Anal. Biochem.72:248-254). Equivalent amounts of protein from each sample wereanalyzed for proteolytic activity on casein zymograms, using well-knownmethods (Mulligan-Kehoe, et al. (2001) supra).

[0108] Identification of plasminogen cleavage products from rPAI-1protein, uPA, and plasminogen reactions. Either rPAI-1₂₃ or rPAI-1_(Δ23)protein (3, 15, 30 nM) was incubated with two-chain uPA (0.5, 0.25,0.05, 0.025 IU) for one hour at 37° C. Two-chain uPA is required forcleavage of plasminogen into plasmin. After one hour, plasminogen (1 IU)was added to the uPA/rPAI-1 reaction mix, and incubated for anadditional one hour at 37° C. The order in which the reactants wereadded was varied, but in each case the incubation time and temperatureremained the same. The permutations that were used were: 1) rPAI-1protein and tcuPA, then plasminogen; 2) tcuPA and plasminogen, thenrPAI-1 protein; 3) rPAI-1 protein, tcuPA, and plasminogen reactedsimultaneously.

[0109] Reaction mixtures containing rPAI-1 protein, uPA and plasminogenwere electrophoresed and transferred to nitrocellulose. The reactionmixtures were then used for immunoblot analysis. Immunoblots were probedwith antibodies specific for different plasminogen epitopes. All primaryantibodies were used at a concentration of 1 μg/ml. Theplasminogen-specific antibodies were raised against either K1-3 (R&DSystems, Inc., Minneapolis, Minn.), K4 (American Diagnostica, Greenwich,Conn.), or mini-plasminogen (American Diagnostica). Mini-plasminogen isdefined by the vendor as K5 plus the serine protease domain. Theantibody to mini-plasminogen does not recognize complexed plasmin. Themembranes were probed for K1-3. The membranes probed for K4 ormini-plasminogen were first incubated with 1 μg/ml of theepitope-specific primary antibodies for one hour at room temperature.Then a rabbit anti-mouse IgG HC+LC (Pierce, Rockford, Ill.) polyclonalantibody at a concentration of 1 μg/ml was incubated with each membranefor 1 hour at room temperature. A horseradish peroxidase-conjugatedantibody (donkey anti-rabbit IgG, Amersham, Arlington Heights, Ill.)amplified the binding reaction which was ultimately detected by additionof a chemiluminescent substrate (Amersham Pharmacia).

[0110] Plasminogen cleavage products in reactions containing rPAI-1 andplasminogen. The rPAI-1₂₃ and rPAI-1_(Δ23) proteins (3, 15, and 30 nM)were each incubated with plasminogen (1 Unit) for two hours at 37° C.The proteins were denatured at 100° C. for five minutes beforeelectrophoresis on a. 10-20% SDS, non-reducing, polyacrylamide gel. Theproteins were transferred to a nitrocellulose membrane and probed formini-plasminogen.

[0111] Binding interactions of rPAI-1 and uPA. Each rPAI-1 protein (3,15, and 30 nM) was incubated with 0.5 IU of either scuPA or tcuPA fortwo hours at 37° C. The reaction mixture was electrophoresed on a 4-20%,SDS, non-reducing polyacrylamide gel. The proteins were transferred tonitrocellulose. The immunoblots were incubated for one hour at roomtemperature with 1 μg/ml of a monoclonal antibody raised against anepitope in the B-chain of uPA near the catalytic domain (AmericanDiagnostica). This antibody recognizes single-chain and two-chain uPA. Asecondary rabbit anti-mouse IgG HC+LC (Pierce, Rockford, Ill.)polyclonal antibody at a concentration of 1 μg/ml was incubated with themembrane for one hour at room temperature. A horseradishperoxidase-conjugated antibody (donkey anti-rabbit IgG, Amersham,Arlington Heights, Ill.) amplified the binding reaction which wasultimately detected by addition of a chemiluminescent substrate(Amersham Pharmacia).

[0112] Apoptosis Detection. Bovine aortic endothelial cells (BAEC) wereplated at a density of 1.0×10⁶ cells/T-75 culture flask containingDulbecco's Modified Eagle's Medium (DMEM) (Gibco BRL, Gaithersburg, Md.)supplemented with 10% fetal bovine serum, penicillin/streptomycin (100IU/ml), and L-glutamine (0.292 mg/ml) (Gibco BRL, Gaithersburg, Md.).The cells were incubated for 24 hours at 37° C., 5% CO₂ before adding 3nM of either exogenous rPAI-1₂₃, rPAI-1_(Δ23), rPAI-1_(Hep23), rPAI-1₂₄or supernatant from P. pastoris culture medium. The cells were incubatedan additional 48 hour at 37° C. Adherent cells were trypsinized,resuspended in DMEM containing 10% FBS, and incubated at 37° C. for 10minutes, then pelleted. The pelleted cells were washed twice with coldHanks' balanced salt solution (HBSS). Fluorescein isothiocyanate (FITC)conjugated APOPNEXIN™ and propidium iodide (PI) were added to the cellsfollowing the manufacturer's protocol (Integren, Purchase,N.Y.)(Koopman, et al. (1994) Proc. Natl. Acad. Sci. USA 84:1415-1420).Each cell fraction was analyzed separately on a FACScan (BectonDickenson, San Jose, Calif.). The data represent five separateexperiments, each performed in duplicate.

[0113] Proliferation. Bovine endothelial cells, treated with exogenousrPAI-1 protein, were plated into 6-well culture plates at a density of1.0×10⁴/ml to assess their proliferative properties in the presence ofrPAI-1 proteins. The cells were trypsinized and counted on ahemocytometer plate at 48 and 96 hours after adding exogenous rPAI-1. Tofurther ascertain the proliferative properties of the rPAI-1-treatedcells, a BrdU labeling assay was performed using a FITC-labeledBrdU-specific antibody. The addition of PI enabled a microscopic countof the proliferating cells relative to the total number of cells. BrdUwas added to the culture medium to obtain a final concentration of 10μM. Cells were incubated for 30 minutes at 37° C. in a CO₂ incubator andthen washed twice in phosphate buffered saline (PBS) containing 1%bovine serum albumin (BSA). FITC-conjugated anti-BrdU was diluted2.5-fold in 0.5% Tween 20/PBS and added directly to the cell culturemedium for 30 minutes at room temperature. The cells were washed in PBSand incubated with PI for 1 minute. Incorporation of BrdU was calculatedby counting the number of cells containing FITC stain (green) or PIstain (red) in 5 fields/sample in triplicate experiments.

[0114] RPAI-1 Effect on Tubule Formation in a Chick Aortic Arch RingAssay. Aortic arches were removed from fertilized chicken eggs (OliverMerrill & Sons, Londonderry, N.H.) at day 14 of embryonic development.The eggs were cracked into a sterile 100 mm culture dish. The embryo wasremoved from its surroundings by cutting away the associated membranesand yolk sac. The chick embryo was placed ventral side up to surgicallyexpose the heart and aortic arches. The heart and aortic arch wereremoved and placed into a sterile culture dish containing PBS to which1% penicillin/streptomycin (Gibco BRL, Gaithersburg, Md.) was added.Arches, from which the surrounding adventitia had been removed, were cutinto 0.8 mm sections. Each arch was placed into 1-5 μl of MATRIGEL™(Kleinman, et al. (1982) Biochemistry 21:6188-6193) that was depositedon the bottom of a 6-well culture plate just prior to adding the ring.An additional 10 μl of ice-cold MATRIGEL™ was spread in a circlesurrounding each aortic arch. The MATRIGEL™ was allowed to solidifybefore adding 2 ml of human endothelial-SFM basal growth medium (GibcoBRL). An rPAI-1 protein and bovine brain extract (BBE)(Clonetics, SanDiego, Calif.), at 30 nM and 10%, respectively were added to each welland incubated at 37° C., 5% CO₂. In order to assess the characteristicsof the new sprouts that could be ascribed to VEGF and thecharacteristics that were inhibited by rPAI-1₂₃, VEGF-A (100 ng/ml) andrPAI-1₂₃ (30 nM) were added to the culture medium containing the aorticrings. At 48 hour, additional medium containing BBE or VEGF and rPAI-1protein was added to the aortic rings. Growth at 37° C. was continuedfor an additional 48 hours. Quantitative evaluation of tubule formationwas performed by a blinded observer on a scale of 1-5 (least to maximumsprouting).

[0115] In experiments with rPAI-1₂₃ and rPAI-1_(Δ23), when cells reachedconfluence, they were placed in separate wells and treated with;rPAI-1₂₃ or rPAI-1_(Δ23) (0.6 nM), rPAI-1₂₃ or rPAI-1_(Δ23) (0.6 nM) and0.25 units of either scuPA or tcuPA; or a reaction containing rPAI-1₂₃or rPAI-1_(Δ23) (0.6 nM) incubated with 0.25 units of either scuPA ortcuPA for one hour at 37° C. Following 24 hours of treatment, theculture medium was removed, the cells were washed in HBSS, and isolatedin 1 ml of cell dissociation buffer (Sigma, St. Louis, Mo.). The viablecells in each sample were counted and 1×10⁵ cells from each sample wereadded to triplicate six-well plates coated with MATRIGEL™. The cellswere incubated for an additional 24 hours at 37° C. Each well wasphotographed with a 35 mm camera at 40× and 200× magnification under aNikon inverted microscope.

EXAMPLE 3 VEGF-A Interactions

[0116] Biochemical Interactions of a VEGF-Heparin Complex with rPAIProteins, uPA, and Plasmin. VEGF was isolated from bovine aorticendothelial cells by first incubating the cells overnight in serum-freeDMEM. The medium was changed before adding 100 μg/ml heparin (Sigma, St.Louis, Mo.) for 4 hours at 37° C. The serum-free DMEM containing theVEGF-heparin complex was precipitated in 80% ethanol. The serum-freemedium from which the VEGF-heparin complex was isolated, was probed forVEGF-A₁₂₁ (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) to insurethat his non-heparin-binding VEGF isoform did not precipitate with theVEGF-heparin complex. VEGF-A₁₂₁ was not detected. The heparin-VEGFcomplex was incubated with rPAI-1₂₃, rPAI-1_(Δ23), rPAI-1_(Hep23), orrPAI-1₂₄ protein (3, 15, and 30 nM) for 2 hours at 37° C. Either uPA(0.25 IU) or plasmin (made from 1.0 IU plasminogen and 0.25 IU uPA) wasadded to the VEGF-heparin-rPAI-1 reaction. After an additional 2 hoursincubation at 37° C. with uPA or plasmin, DTT, at a final concentrationof 0.1 M, was added to one half of the reaction mixture for 3.5 hours at37° C. An equal volume of each reaction mixture was denatured at 95° C.,then electrophoresed on a 4-20% SDS polyacrylamide gel. The VGEF in eachsample was visualized on a nitrocellulose membrane probed withmonoclonal antibody to human VEGF-A_(165,189,206) (BD PharMingen, SanDiego, Calif.). A secondary rabbit anti-mouse IgG HC+LC polyclonalantibody (Pierce, Rockford, Ill.) was diluted to 1 μg/ml in TBS, pH 8.0,containing 5% skim milk, and incubated with anti-VEGF probed membranesfor 1 hour at room temperature. A horseradish peroxidase-conjugatedantibody (donkey anti-rabbit IgG, Amersham, Arlington Heights, Ill.)diluted 1:500 amplified the binding reaction which was ultimatelydetected by addition of a chemiluminescent substrate (Amersham,Arlington Heights, Ill.).

[0117] VEGF-A in the Culture Medium of rPAI-1-Treated Endothelial Cells.Bovine aortic endothelial cells were seeded into six-well culture platesand grown to confluence in DMEM. The confluent (quiescent) cells weretreated with a single dose of rPAI-1 protein at a final concentration of1.2 nM. At 6, 12, 15, 24, 30, 48, and 72 hours after the onset oftreatment, the culture medium was removed from cells treated with eachrPAI-1 protein. Equivalent amounts of protein were incubated with 0.1 MDTT for 2.5 hours at 37° C. The protein samples were electrophoresed ona 4-20% gradient, SDS-polyacrylamide gel. The proteins were transferredto a nitrocellulose membrane and probed for VEGF-A_(165,189,206).

[0118] VEGF-A Bound to Heparan Sulfate in rPAI-1-treated EndothelialCells. Bovine aortic endothelial cells were seeded into six-well cultureplates and treated with rPAI-1 proteins. Following a 6-and 12-hourincubation with the rPAI-1 proteins, the culture medium was removed, thecell layer was washed twice in HBSS. The cells were incubated for 1 hourat 37° C. in 1 ml of HBSS-containing 0.05 IU of heparinase III (Sigma,St. Louis, Mo.). Following the incubation, the HBSS-containing proteinsreleased during the enzymatic digest were collected. The proteins wereconcentrated in 80% ethanol. An equivalent amount of protein from eachsample was incubated with 0.1 M DTT at 37° C. for 2.5 hours prior toelectrophoresis in a 4-20% gradient SDS-polyacrylamide gel. Theseparated proteins were transferred to an immunoblot and probed forVEGF-A using an antibody specific for epitopes common toVEGF-A_(165,189,209) (BD Pharmingen, San Diego, Calif.). In anotherseries of experiments the immunoblots were probed simultaneously withtwo competing antibodies to VEGF-A; one antibody was specific for theactive site of VEGF-A₁₆₅ (R&D Systems, Inc., Minneapolis, Minn.) and thesecond antibody was raised against an epitope common toVEGF-A_(165,189,206). The binding reactions occurred at 4° C. for 15hours. The binding reaction was further amplified, as described herein.The chemiluminescent detection was performed, as described herein.

EXAMPLE 4 Apoptosis in rPAI-1₂₃-Treated BAEC vs PAEC

[0119] Bovine aortic endothelial cells (BAEC) and porcine aorticendothelial cells (PAEC) were selected for comparison of apoptosis basedon the expression of VEGFR-1 and VEGFR-2. BAEC express both receptorsand PAEC does not express either receptor (Gille, et al. (2001) J. Biol.Chem. 276:3222-3230). BAEC and PAEC were seeded into T-25 flaskscontaining DMEM supplemented with 10% fetal bovine serum,penicillin/streptomycin (100 IU/ml), and L-glutamine (0.292 mg/ml). Thecells were incubated at 37° C., 5% CO₂ until the cells reachedconfluence, at which time fresh culture medium containing 2.4 mMrPAI-1₂₃ was added to the cells. The treated cells continued to grow foran additional 36 hours before harvesting for analysis of apoptosis in anAnnexin V assay.

EXAMPLE 5 Growth Factor-Independent Activity of rPAI-1_(Hep23)

[0120] Ex vivo Assay. The aortic arch was surgically removed from14-day-old chick embryos, and sectioned into rings of approximately 0.8mm. The rings were placed on MATRIGEL™ to promote tubule formation inendothelial-SFM basal growth medium containing 10% fetal bovine serumand either bFGF/VEGF (each at 50 ng/ml) or rPAI-1_(Hep23) (15 nM).Growth of tubules was at 37° C., 5% CO₂ for 4 days. At day 2, additionalmedium containing growth factors or rPAI-1_(Hep23) was added.

[0121] In vivo Assay. The rPAI-1₂₃ protein, at a concentration of 20μg/ml, was added to 0.5 ml of MATRIGEL™ containing 25 ng/ml of VEGF andbFGF. The rPAI-1_(Hep23) protein (20 μg/ml) was added to the MATRIGEL™in the absence of bFGF/VEGF. The mixtures were kept on ice. Each testsample was injected into the right flank of eight C3H female mice.Neoangiogenic vessels were allowed to form around and into the MATRIGEL™plug for two weeks. At the end of the test period, the MATRIGEL™ pelletswere surgically removed from the animals and fixed in 2.5%paraformaldehyde. The fixed pellets were embedded in paraffin from whichsections were cut. Neoangiogenic vessels were counted in five sectionsprepared from each pellet.

1 8 1 27 PRT Sus scrofa 1 Met Gln Phe Lys Ile Glu Glu Lys Gly Met AlaPro Ala Leu Arg Gln 1 5 10 15 Leu Tyr Lys Glu Leu Met Gly Pro Trp AsnLys 20 25 2 24 PRT Sus scrofa 2 Lys Ile Glu Glu Lys Gly Met Ala Pro AlaLeu Arg Gln Leu Tyr Lys 1 5 10 15 Glu Leu Met Gly Pro Trp Asn Lys 20 326 DNA Artificial Sequence Synthetic oligonucleotide primer. 3ggaattcatg gatgagatca gcacgg 26 4 24 DNA Artificial Sequence Syntheticoligonucleotide primer. 4 gctctagatt tccactggct gatg 24 5 34 DNAArtificial Sequence Synthetic oligonucleotide primer. 5 ggaattcatgcagttcaaga ttgaggagaa gggc 34 6 24 DNA Artificial Sequence Syntheticoligonucleotide primer. 6 gctctagatt tccactggct gatg 24 7 20 DNAArtificial Sequence Synthetic oligonucleotide primer. 7 ggaattcaaggagctcatgg 20 8 23 DNA Artificial Sequence Synthetic oligonucleotideprimer. 8 gctctagatc aaggctccat cac 23

What is claimed is:
 1. A method for producing a 34 kDa truncated plasminproteolytic protein comprising combining plasminogen and rPAI-1₂₃ for aspecified amount of time and adding uPA so that a 34 kDa truncatedplasmin proteolytic protein is produced.
 2. A 34 kDa truncated plasminproteolytic protein produced by the method of claim
 1. 3. A method formodulating the expression of a membrane type 1-matrix metalloproteinasecomprising administering an effective amount of an plasminogen activatorinhibitor type 1 isoform lacking a reactive center loop and containing acomplete heparin-binding domain or lacking at least a portion of aheparin-binding domain so that the expression of a membrane type1-matrix metalloproteinase is modulated.
 4. The method of claim 3,wherein the plasminogen activator inhibitor type isoform contains theheparin-binding domain and increases expression of a membrane type1-matrix metalloproteinase.
 5. The method of claim 3, wherein theplasminogen activator inhibitor type isoform lacks a portion of theheparin-binding domain and decreases expression of a membrane type1-matrix metalloproteinase.